Alpha-amylase variants with altered 1, 6-activity

ABSTRACT

The present invention relates to variants of parent Termamyl-like alpha-amylases, which variant has alpha-amylase activity and exhibits an alteration in the alpha-1,6-D-glucosidic branch linkage cleavage activity of amylopectin and limit dextrins.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims, under 35 U.S.C. 119, priority of Danishapplication Ser. No. PA 2000 00779 filed May 12, 2000, and the benefitof U.S. provisional application No. 60/205,229, filed May 17, 2000, thecontents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to variants (mutants) of parentTermamyl-like alpha-amylases, which variant exhibits an alteration inalpha-1,6-D-glucosidic branch linkage cleavage activity. The inventionalso relates a DNA construct comprising a DNA sequence encoding thealpha-amylase variant of the invention, an expression vector forrecombinant production and a host cell for recombinant production. Theinvention also relates to various compositions comprising a variant ofthe invention, especially for use in the liquefaction step of, e.g., astrach convention or ethanol process, and finally to the use of suchvariants or compositions of the invention for various uses.

BACKGROUND OF THE INVENTION

[0003] Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C.3.2.1.1) constitute a group of enzymes, which catalyze hydrolysis ofstarch and other linear and branched 1,4-glucosidic oligo-andpolysaccharides.

[0004] Protein engineering is increasingly used to alter the propertiesof enzymes—such as alpha-amylase-to obtain an enzyme with propertiestailored for specific applications.

BRIEF DISCLOSURE OF THE INVENTION

[0005] The object of the present invention is to provide Termamyl-likeamylases which variants in comparison to the corresponding parentalpha-amylase, i.e., un-mutated alpha-amylase, has alpha-amylaseactivity and exhibits an alteration in alpha-1,6-D-glucosidic branchlinkage cleavage activity of amylopectin and alpha- and beta-limitdextrins.

[0006] Alpha-amylases with altered activity towardsalpha-1,6-D-glucosidic branch linkages of amylopectin and alpha- andbeta-limit dextrins are desired, because such enzyme variants may, ifthe 1,6-activity is increased, increase the glucose yield in theliquefaction process in connection with, e.g., high fructose corn syrup(HFCS) production, because less panose will be formed. Further, indetergents the degradation of starch into smaller sugar units (lesslimit dextrins or smaller limit dextrins) facilitates the washing out ofthe sugar molecules and thus improve the wash performance. If a higherlimit dextrins level is desired reduced 1,6-activity is advantageous.

Nomenclature

[0007] In the present description and claims, the conventionalone-letter and three-letter codes for amino acid residues are used. Forease of reference, alpha-amylase variants of the invention are describedby use of the following nomenclature:

[0008] Original amino acid(s): position(s): substituted amino acid(s)

[0009] According to this nomenclature, for instance the substitution ofalanine for asparagine in position 30 is shown as:

[0010] Ala30Asn or A30N

[0011] a deletion of alanine in the same position is shown as:

[0012] Ala30* or A30*

[0013] and insertion of an additional amino acid residue, such aslysine, is shown as:

[0014] Ala30AlaLys or A30AK

[0015] A deletion of a consecutive stretch of amino acid residues, suchas amino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33).

[0016] Where a specific alpha-amylase contains a “deletion” incomparison with other alpha-amylases and an insertion is made in such aposition this is indicated as:

[0017] *36Asp or *36D for insertion of an aspartic acid in position 36.Multiple mutations are separated by plus signs, i.e.:

[0018] Ala30Asp+Glu34Ser or A30N+E34S representing mutations inpositions 30 and 34 substituting alanine and glutamic acid forasparagine and serine, respectively.

[0019] When one or more alternative amino acid residues may be insertedin a given position it is indicated as

[0020] A30N,E or

[0021] A30N or A30E

[0022] Furthermore, when a position suitable for modification isidentified herein without any specific modification being suggested, itis to be understood that any amino acid residue may be substituted forthe amino acid residue present in the position. Thus, for instance, whena modification of an alanine in position 30 is mentioned, but notspecified, it is to be understood that the alanine may be deleted orsubstituted for any other amino acid, i.e., any one of:

[0023] R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

[0024] Further, “A30X” means any one of the following substitutions:A30R, A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A30I, A30L, A30K, A30M,A30F, A30P, A30S, A30T, A30W, A30Y, or A30 V; or in short:A30R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

[0025] In the first aspect the invention relates a variant of a parentTermamyl-like alpha-amylase, comprising an alteration at one or more ofthe following regions or positions selected from the group of:

[0026] Region: 186-193,

[0027] Region: 261-276,

[0028] Region: 283-293,

[0029] Region: 334-339,

[0030] Position: 234, wherein

[0031] (a) the alteration(s) are independently

[0032] (i) an insertion of an amino acid downstream of the amino acidwhich occupies the position,

[0033] (ii) a deletion of the amino acid which occupies the position, or

[0034] (iii) a substitution of the amino acid which occupies theposition with a different amino acid,

[0035] (b) the variant has alpha-amylase activity and (c) each positioncorresponds to a position of the amino acid sequence of the parentTermamyl-like alpha-amylase having the B. licheniformis alpha-amylaseamino acid sequence of shown in SEQ ID NO: 8.

[0036] The invention also relates to a number of specific variants; to aDNA construct comprising a DNA sequence encoding an alpha-amylasevariant of the invention; a recombinant expression vector which carriesa DNA construct of the invention; a cell which is transformed with a DNAconstruct of the invention or a vector of the invention; compositioncomprising alpha-amylase variant of the invention; the use of a variantof the invention for specified industrial applications.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037]FIG. 1 is an alignment of the amino acid sequences of five parentTermamyl-like alpha-amylases. The numbers on the extreme left designatethe respective amino acid sequences as follows:

[0038] 1: SEQ ID NO: 2

[0039] 2: SEQ ID NO: 1

[0040] 3: SEQ ID NO: 5

[0041] 4: SEQ ID NO: 4

[0042] 5: SEQ ID NO: 3.

DETAILED DISCLOSURE OF THE INVENTION

[0043] The object of the present invention is to provide Termamyl-likeamylases, which variants exhibits an alteration in thealpha-1,6-D-glucosidic branch linkage cleavage activity of especiallyamylopectin and alpha—and/or beta—limit dextrins.

Altered Alpha-1,6-D-Glucosidic Branch Linkage Activity

[0044] Alpha-amylases, which catalyze hydrolysis of starch and otherlinear and branched 1,4-glucosidic oligo—and polysaccharides have ingeneral limited activity on the alpha-1,6-D-glucosidic branch linkagesof amylopectin and alpha- and beta-limit dextrins.

[0045] For instance, to increase the final dextrose yield in starchconvention processes it is desirable to break the alpha-1,6-D-glucosidicbranch linkages in amylopectin and alpha—and/or beta-limit dextrins.

[0046] If an increased amount of alpha—and/or beta-limit dextrins isdesired it may be advantageous to decrease the alpha-1,6-D-glucosidicbranch linkages activity.

[0047] The present inventors have found that the alpha-1,6-D-glucosidicbranch linkages of amylopectin and/or alpha—and/or beta-limit dextrinscan be altered by mutating within one or more of the below mentionedregions or positions in Termamyl-like alpha-amylases.

[0048] Thus, in the first aspect the invention relates a variant of aparent Termamyl-like alpha-amylase, comprising an alteration at one ormore of the following regions or positions selected from the group of:

[0049] Region: 186-193,

[0050] Region: 261-276,

[0051] Region: 283-293,

[0052] Region: 334-339,

[0053] Position 234,

[0054] wherein

[0055] (a) the alteration(s) are independently

[0056] (i) an insertion of an amino acid downstream of the amino acidwhich occupies the position,

[0057] (ii) a deletion of the amino acid which occupies the position, or

[0058] (iii) a substitution of the amino acid which occupies theposition with a different amino acid,

[0059] (b) the variant has alpha-amylase activity and (c) each positioncorresponds to a position of the amino acid sequence of the parentTermamyl-like alpha-amylase having the B. licheniformis alpha-amylaseamino acid sequence of shown in SEQ ID NO: 8.

[0060] In an embodiment the region mutated is the Region: 186-193.Specific preferred positions contemplated include one or more ofpositions 186, 187, 188, 189, 190, 191, 192, 193. Specific mutationsinclude one or more of: E189GASTV;

[0061] In another embodiment the region mutated is the Region: 261-276.Specific preferred positions contemplated include one or more ofpositions 261, 262, 263, 264, 265, 266, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276. Specific mutations include one or more of:

[0062] W263GASTV; Q264X; N265GASTV;

[0063] In another embodiment the region mutated is the Region: 283-293.Specific preferred positions contemplated include one or more ofpositions 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293.Specific mutations include one or more of: V286FWY or smaller residuesthan V, e.g., GAS; Y290 smaller residues than Y, e.g.,A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,V.

[0064] In another embodiment the region mutated is the Region: 334-339.Specific preferred positions contemplated include one or more ofpositions 334, 335, 336, 337, 338, 339. Specific mutations include oneor more of: L335G,A,S,T,V;

[0065] Finally, position K234 is also contemplated according to theinvention, preferably K234X, especially preferably K234N,Q.

Detection of 1,6-Activity of Alpha-Amylases

[0066] In the presence of 1,4-activity, additional 1,6-activity can bemeasured by NMR spectroscopic methods. ¹H NMR spectroscopy allows adirect measure of the relative amounts of the anomeric protons ofalpha(1→4)-linkages, alpha(1→6)-linkages and reducing ends,respectively. With a suitable substrate the hydrolysis of the individualbond types can be followed during reaction, e.g., hydrolysis ofalpha(1→4)-linkages will give a reduction in the integral of thealpha(1→4)-signals and an equivalent increase in the integral of thesignals coming from the reducing ends. In case of an enzyme, which havea supplementary 1,6-activity it is useful to look at the ratioR—(I_(alpha)(1→4)+I_(red. ends))/I_(alpha)(1→6), where I is theintegral. If only alpha(1→4)-linkages are being hydrolysed R will remainconstant throughout the reaction. If alpha(1→6)-linkages are also beingcleaved R will increase.

[0067] Synthetic designed substrates and limit dextrins can be used assubstrates. Termamyl® (B. licehniformis alpha-amylase shown in SEQ IDNO: 8) and Novamyl® (B. stearothermophilus C599 maltogenic amylasedisclosed in EP patent no. 120,693 (Novo Industri A/S) limit dextrins(made from amylopectin), where the low molecular weight material (<1000Da) has been removed by ultrafiltration may be used. The incubations canbe followed directly in the NMR tube (D₂O) at 60° C. if enzyme andsubstrate are pretreated repeatedly with D₂O.

[0068] An NMR procedure for detection of 1,6 activity is described belowin “Materials & Methods” section.

[0069] Limit dextrins may be prepared as described by Mottawia et al,Carbohydr. Res. 277 (1995), 109-123 (which is hereby incorporated byreference) or specifically as described in the “Materials & Methods”section.

[0070] A variant of the invention may in an embodiment be capable ofhydrolysing starch and other linear and branched 1,4-glucosidicoligo—and polysaccharides and may further have altered (i.e., higher orlower) alpha-1,6-D-glucosidic branch linkage cleavage activity.

[0071] In another embodiment the variant of the invention is capable ofhydrolysing starch and other linear and branched 1,4-glucosidicoligo—and polysaccharides, but do not have any detectablealpha-1,6-D-glucosidic branch linkage cleavage activity.

Termamyvl-like Alpha-Amylases

[0072] A number of alpha-amylases produced by Bacillus spp. are highlyhomologous (identical) on amino acid level. The identity of a number ofBacillus alpha-amylases can be found in the below Table 1: TABLE 1Percent identity 707 AP1378 BAN BSG SP690 SP722 AA560 Termamyl 707 100.086.4 66.9 66.5 87.6 86.2 95.5 68.1 AP1378 86.4 100.0 67.1 68.1 95.1 86.686.0 69.4 BAN 66.9 67.1 100.0 65.6 67.1 68.8 66.9 80.7 BSG 66.5 68.165.6 100.0 67.9 67.1 66.3 65.4 SP690 87.6 95.1 67.1 67.9 100.0 87.2 87.069.2 SP722 86.2 86.6 68.8 67.1 87.2 100.0 86.8 70.8 AA560 95.5 86.0 66.966.3 87.0 86.8 100.0 68.3 Termamyl 68.1 69.4 80.7 65.4 69.2 70.8 68.3100.0

[0073] For instance, the B. licheniformis alpha-amylase comprising theamino acid sequence shown in SEQ ID NO: 8 (commercially available asTermamyl™) has been found to be about 81% homologous (identical) to theB. amyloliquefaciens alpha-amylase comprising the amino acid sequenceshown in SEQ ID NO: 10 and about 65% homologous with the B.stearothermophilus alpha-amylase comprising the amino acid sequenceshown in SEQ ID NO: 6. Further homologous parent alpha-amylases includeSP690 and SP722, respectively, disclosed in WO 95/26397 and furtherdepicted in SEQ ID NO: 2 and SEQ ID NO: 4, respectively, herein. OtherTermamyl-like alpha-amylases are the AA560 alpha-amylase derived fromBacillus sp. and shown in SEQ ID NO: 12, and the #707 alpha-amylasederived from Bacillus sp., shown in SEQ ID NO: 13 and described byTsukamoto et al., Biochemical and Biophysical Research Communications,151 (1988), pp. 25-31. The Termamyl-like alpha-amylase referred to areKSM AP1378 is disclosed in WO 97/00324 (from KAO Corporation, JP).

[0074] Still further homologous Termamyl-like alpha-amylases include thealpha-amylase produced by the B. licheniformis strain described in EP0252666 (ATCC 27811), and the alpha-amylases identified in WO 91/00353and WO 94/18314. Other commercially available Termamyl-likealpha-amylases are comprised in the products sold under the followingtradenames: Optitherm™ and Takatherm™ (available from Solvay); Maxamyl™(available from Gist-brocades(DSM)/Genencor), Spezym AA™ and SpezymeDelta AA™ (available from Genencor), and Keistase™ (available fromDaiwa), PURASTAR

ST 5000E, PURASTRAR

HPAM L (from Genencor Int.).

[0075] Because of the substantial homology found between thesealpha-amylases, they are considered to belong to the same class ofalpha-amylases, namely the above-mentioned class of “Termamyl-likealpha-amylases”.

[0076] Accordingly, in the present context, the term “Termamyl-likealpha-amylase” is intended to indicate an alpha-amylase, which, at theamino acid level, exhibits a substantial identity to Termamyl™, i.e.,the B. licheniformis apha-amylase having the amino acid sequence shownin SEQ ID NO: 8 herein. In other words, all the followingalpha-amylases, which has the amino acid sequences shown in SEQ ID NOS:2, 4, 6, 8, 10, 12 and 13 herein are considered to be “Termamyl-likealpha-amylase”. Other Termamyl-like alpha-amylases are alpha-amylases i)which displays at least 60%, such as at least 70%, e.g., at least 75%,or at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,at least 98%, at least 99% homology (identity) with at least one of theamino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, and 13,and/or ii) is encoded by a DNA sequence which hybridizes to the DNAsequences encoding the above-specified alpha-amylases which are apparentfrom SEQ ID NOS: 1, 3, 5, 7, 9, and of the present specification (whichencoding sequences encode the amino acid sequences shown in SEQ ID NOS:2, 4, 6, 8, 10 and 12 herein, respectively)

[0077] In connection with property i), the term “homology” may bedetermined as the degree of “identity” between the two sequencesindicating a derivation of the first sequence from the second. Thehomology (identity) may suitably be determined by means of computerprograms known in the art such as GAP provided in the GCG programpackage (described above). Thus, Gap GCGv8 may be used with thefollowing default parameters: GAP creation penalty of 5.0 and GAPextension penalty of 0.3, default scoring matrix, for nucleic sequencesand 3.0 and 0.1, respectively, from protein sequences. GAP uses themethod of Needleman/Wunsch/Sellers to make alignments.

[0078] A structural alignment between, e.g., Termamyl® (SEQ ID NO: 8)and a Termamyl-like alpha-amylase may be used to identifyequivalent/corresponding positions in other Termamyl-likealpha-amylases. One method of obtaining said structural alignment is touse the Pile Up programme from the GCG package using default values ofgap penalties, i.e., a gap creation penalty of 3.0 and gap extensionpenalty of 0.1. Other structural alignment methods include thehydrophobic cluster analysis (Gaboriaud et al., (1987), FEBS LETTERS224, pp. 149-155) and reverse threading (Huber, T; Torda, AE, PROTEINSCIENCE Vol. 7, No. 1 pp. 142-149 (1998).

Hybridisation

[0079] The oligonucleotide probe used in the characterisation of theTermamyl-like alpha-amylase in accordance with property ii) above maysuitably be prepared on the basis of the full or partial nucleotide oramino acid sequence of the alpha-amylase in question.

[0080] Suitable conditions for testing hybridisation involve pre-soakingin 5xSSC and prehybridizing for 1 hour at ^(˜)40° C. in a solution of20% formamide, 5xDenhardt's solution, 50 mM sodium phosphate, pH 6.8,and 50 mg of denatured sonicated calf thymus DNA, followed byhybridisation in the same solution supplemented with 100mM ATP for 18hours at ^(˜)40° C., followed by three times washing of the filter in2xSSC, 0.2% SDS at 40° C. for 30 minutes (low stringency), preferred at50° .C (medium stringency), more preferably at 65° C. (high stringency),even more preferably at ^(˜)75° C. (very high stringency). More detailsabout the hybridisation method can be found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989.

[0081] In the present context, “derived from” is intended not only toindicate an alpha-amylase produced or producible by a strain of theorganism in question, but also an alpha-amylase encoded by a DNAsequence isolated from such strain and produced in a host organismtransformed with said DNA sequence. Finally, the term is intended toindicate an alpha-amylase, which is encoded by a DNA sequence ofsynthetic and/or cDNA origin and which has the identifyingcharacteristics of the alpha-amylase in question. The term is alsointended to indicate that the parent alpha-amylase may be a variant of anaturally occurring alpha-amylase, i.e., a variant, which is the resultof a modification (insertion, substitution, deletion) of one or moreamino acid residues of the naturally occurring alpha-amylase.

Parent Termamyl-like Alpha-Amylases

[0082] According to the invention all Termamy-like alpha-amylases, asdefined above, may be used as the parent (i.e., backbone) alpha-amylase.In a preferred embodiment of the invention the parent alpha-amylase isderived from B. lichenifo-rmis, e.g., one of those referred to above,such as the B. licheniformis alpha-amylase having the amino acidsequence shown in SEQ ID NO: 8.

Parent Hybrid Termamyl-Like Alpha-Amylases

[0083] The parent alpha-amylase (i.e., backbone alpha-amylase) may alsobe a hybrid alpha-amylase, i.e., an alpha-amylase, which comprises acombination of partial amino acid sequences derived from at least twoalpha-amylases.

[0084] The parent hybrid alpha-amylase may be one, which on the basis ofamino acid homology (identity) and/or DNA hybridization (as definedabove) can be determined to belong to the Termamyl-like alpha-amylasefamily. In this case, the hybrid alpha-amylase is typically composed ofat least one part of a Termamyl-like alpha-amylase and part(s) of one ormore other alpha-amylases selected from Termamyl-like alpha-amylases ornon-Termamyl-like alpha-amylases of microbial (bacterial or fungal)and/or mammalian origin.

[0085] Thus, the parent hybrid alpha-amylase may comprise a combinationof partial amino acid sequences deriving from at least two Termamyl-likealpha-amylases, or from at least one Termamyl-like and at least onenon-Termamyl-like bacterial alpha-amylase, or from at least oneTermamyl-like and at least one fungal alpha-amylase. The Termamyl-likealpha-amylase from which a partial amino acid sequence derives, may beany of those specific Termamyl-like alpha-amylase referred to herein.

[0086] For instance, the parent alpha-amylase may comprise a C-terminalpart of an alpha-amylase derived from a strain of B. licheniformis, anda N-terminal part of an alpha-amylase derived from a strain of B.amyloliquefaciens or from a strain of B. stearothermophilus. Forinstance, the parent alpha-amylase may comprise at least 430 amino acidresidues of the C-terminal part of the B. licheniformis alpha-amylase,and may, e.g., comprise a) an amino acid segment corresponding to the 37N-terminal amino acid residues of the B. amyloliquefaciens alpha-amylasehaving the amino acid sequence shown in SEQ ID NO: 10 and an amino acidsegment corresponding to the 445 C-terminal amino acid residues of theB. licheniformis alpha-amylase having the amino acid sequence shown inSEQ ID NO: 8, or a hybrid Termamyl-like alpha-amylase being identical tothe Termamyl sequence, i.e., the Bacillus licheniformis alpha-amylase(BLA) shown in SEQ ID NO: 8, except that the N-terminal 35 amino acidresidues (of the mature protein) has been replaced by the N-terminal 33residues of BAN (mature protein), i.e., the Bacillus amyloliquefaciensalpha-amylase (BAN) shown in SEQ ID NO: 10; or b) an amino acid segmentcorresponding to the 68 N-terminal amino acid residues of the B.stearothermophilus alpha-amylase (BSG) having the amino acid sequenceshown in SEQ ID NO: 6 and an amino acid segment corresponding to the 415C-terminal amino acid residues of the B. licheniformis alpha-amylasehaving the amino acid sequence shown in SEQ ID NO: 8.

[0087] In a preferred embodiment of the invention the parentTermamyl-like alpha-amylase is a hybrid alpha-amylase of SEQ ID NO: 8and SEQ ID NO: 10. Specifically, the parent hybrid Termamyl-likealpha-amylase may be a hybrid alpha-amylase comprising the 445C-terminal amino acid residues of the B. licheniformis alpha-amylaseshown in SEQ ID NO: 4 and the 37 N-terminal amino acid residues of thealpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 5,which may suitably further have the following mutations:H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 8). Thelatter mentioned hybrid is referred to as LE174.

[0088] Another suitable parent hybrid alpha-amylase is the onepreviously described in WO 96/23874 (from Novozymes) constituting theN-terminus of BAN, Bacillus amyloliquefaciens alpha-amylase (amino acids1-300 of the mature protein) and the C-terminus from Termamyl® (aminoacids 301-483 of the mature protein). Other specifically contemplatedparent alpha-amylase include LE174 with fewer mutations, i.e., the rightabove mentioned hydrid having the following mutations:A181T+N190F+A209V+Q264S; N190F+A209V+Q264S; A209V+Q264S; Q264S;H156Y+N190F+A209V+Q264S; H156Y+A209V+Q264S; H156Y+Q264S;H156Y+A181T+A209V+Q264S; H156Y+A181T+Q264S; H156Y+Q264S;H156Y+A181T+N190F+Q264S; H156Y+A181T+N190F; H156Y+A181T+N190F+A209V.These hybrids are also considered to be part of the invention.

[0089] In a preferred embodiment the parent Termamyl-like alpha amylaseis LE174, SP722, or AA560 including any of LE174+G48A+T49I+G107A+I201F;LE174+M197L; LE174+G48A+T49I+G107A+M197L+I201F, or SP722+D183*+G184*;SP722+D183*+G184*+N195F; SP722+D183*+G184*+M202L;SP722+D183*+G184*+N195F+M202L; BSG+I181*+G182*; BSG+I181*+G182*+N193F;BSG+Il81*+G182*+M20OL; BSG+I181*+G182*+N193F+M200L; AA560+D183*+G184*;AA560+D183*+G184*+N195F; AA560+D183*+G184*+M202L;AA560+D183*+G184*+N195F+M202L.

General mutations in variants of the invention

[0090] The particularly interesting amino acid substitutions are thosethat increase the mobility around the active site of the enzyme. This isaccomplished by changes that disrupt stabilizing interaction in thevicinity of the active site, i.e., within preferably 10Å or 8Å or 6Å or4Å from any of the residues constituting the active site.

[0091] Examples are mutations that reduce the size of side chains, suchas

[0092] Ala to Gly,

[0093] s Val to Ala or Gly,

[0094] Ile or Leu to Val, Ala, or Gly

[0095] Thr to Ser

[0096] Such mutations are expected to cause increased flexibility in theactive site region either by the introduction of cavities or by thestructural rearrangements that fill the space left by the mutation.

[0097] It may be preferred that a variant of the invention comprises oneor more modifications in addition to those outlined above. Thus, it maybe advantageous that one or more Proline residues present in the part ofthe alpha-amylase variant which is modified is/are replaced with anon-Proline residue which may be any of the possible, naturallyoccurring non-Proline residues, and which preferably is an Alanine,Glycine, Serine, Threonine, Valine or Leucine.

[0098] Analogously, it may be preferred that one or more Cysteineresidues present among the amino acid residues with which the parentalpha-amylase is modified is/are replaced with a non-Cysteine residuesuch as Serine, Alanine, Threonine, Glycine, Valine or Leucine.

[0099] Furthermore, a variant of the invention may—either as the onlymodification or in combination with any of the above outlinedmodifications—be modified so that one or more Asp and/or Glu present inan amino acid fragment corresponding to the amino acid fragment 185-209of SEQ ID NO: 10 is replaced by an Asn and/or Gln, respectively. Also ofinterest is the replacement, in the Termamyl-like alpha-amylase, of oneor more of the Lys residues present in an amino acid fragmentcorresponding to the amino acid fragment 185-209 of SEQ ID NO: 10 by anArg.

[0100] It will be understood that the present invention encompassesvariants incorporating two or more of the above outlined modifications.

[0101] Furthermore, it may be advantageous to introduce furthermutations the variant of the invention. Contemplated are one or more ofthe following positions (using SEQ ID NO: 8 (Termamyl®) for thenumbering):

[0102] M15, V128, A111, H133, W138, T149, M197, N188, A209, A210, H405,T412, in particular the following single, double or triple or multimutations:

[0103] M15X, in particular M15T,L;

[0104] V128X, in particular V128E;

[0105] H133X, in particular H133Y;

[0106] N188X, in particular N188S,T,P;

[0107] M197X, in particular M197T,L;

[0108] A209X, in particular A209V;

[0109] M197T/W138F; M197T/W138Y; M15T/H133Y/N188S;

[0110] M15/V128E/H133Y/N188S; E119C/S130C; D124C/R127C; H133Y/T149I;

[0111] G475R, H133Y/S187D; H133Y/A209V.

Methods for Preparing Alpha-Amylase Variants of the Invention

[0112] Several methods for introducing mutations into genes are known inthe art. After a brief discussion of the cloning ofalpha-amylase-encoding DNA sequences, methods for generating mutationsat specific sites within the alpha-amylase-encoding sequence will bediscussed.

Cloning a DNA Sequence Encoding an Alpha-Amylase

[0113] The DNA sequence encoding a parent alpha-amylase may be isolatedfrom any cell or microorganism producing the alpha-amylase in question,using various methods well known in the art. First, a genomic DNA and/orcDNA library should be constructed using chromosomal DNA or messengerRNA from the organism that produces the alpha-amylase to be studied.Then, if the amino acid sequence of the alpha-amylase is known,homologous, labeled oli-gonucleotide probes may be synthesized and usedto identify alpha-amylase-encoding clones from a genomic libraryprepared from the organism in question. Alternatively, a labeledoligonucleotide probe containing sequences homologous to a knownalpha-amylase gene could be used as a probe to identifyalpha-amylase-encoding clones, using hybridization and washingconditions of lower stringency.

[0114] Yet another method for identifying _(α)-amylase-encoding cloneswould involve inserting fragments of genomic DNA into an expressionvector, such as a plasmid, transforming _(α)-amylase-negative bacteriawith the resulting genomic DNA library, and then plating the transformedbacteria onto agar containing a substrate for alpha-amylase, therebyallowing clones expressing the alpha-amylase to be identified.

[0115] Alternatively, the DNA sequence encoding the enzyme may beprepared synthetically by established standard methods, e.g., thephosphoroamidite method described by S. L. Beaucage and M. H. Caruthers,Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described byMatthes et al., The EMBO J. 3, 1984, pp. 801-805. In thephosphoroamidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

[0116] Finally, the DNA sequence may be of mixed genomic and syntheticorigin, mixed synthetic and cDNA origin or mixed genomic and cDNAorigin, prepared by ligating fragments of synthetic, genomic or cDNAorigin (as appropriate, the fragments corresponding to various parts ofthe entire DNA sequence), in accordance with standard techniques. TheDNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in US 4,683,202 orR.K. Saiki et al., Science 239, 1988, pp. 487-491.

Site-Directed Mutagenesis

[0117] Once an alpha-amylase-encoding DNA sequence has been isolated,and desirable sites for mutation identified, mutations may be introducedusing synthetic oligonucleotides. These oligonucleotides containnucleotide sequences flanking the desired mutation sites; mutantnucleotides are inserted during oligonucleotide synthesis. In a specificmethod, a single-stranded gap of DNA, bridging thealpha-amylase-encoding sequence, is created in a vector carrying thealpha-amylase gene. Then the synthetic nucleotide, bearing the desiredmutation, is annealed to a homologous portion of the single-strandedDNA. The remaining gap is then filled in with DNA polymerase I (Klenowfragment) and the construct is ligated using T4 ligase. A specificexample of this is method is described in Morinaga et al. (1984). US4,760,025 disclose the introduction of oligonucleotides encodingmultiple mutations by performing minor alterations of the cassette.However, an even greater variety of mutations can be introduced at anyone time by the Morinaga method, because a multitude ofoligonucleotides, of various lengths, can be introduced.

[0118] Another method for introducing mutations intoalpha-amylase-encoding DNA sequences is described in Nelson and Long(1989). It involves the 3-step generation of a PCR fragment containingthe desired mutation introduced by using a chemically synthesized DNAstrand as one of the primers in the PCR reactions. From thePCR-generated fragment, a DNA fragment carrying the mutation may beisolated by cleavage with restriction endonucleases and reinserted intoan expression plasmid.

Random Mutagenesis

[0119] Random mutagenesis is suitably performed either as localised orregion-specific random mutagenesis in at least three parts of the genetranslating to the amino acid sequence shown in question, or within thewhole gene.

[0120] The random mutagenesis of a DNA sequence encoding a parent_(α)-amylase may be conveniently performed by use of any method known inthe art.

[0121] In relation to the above, a further aspect of the presentinvention relates to a method for generating a variant of a parentalpha-amylase, e.g., wherein the variant exhibits altered or increasedthermal stability relative to the parent, the method comprising:

[0122] (a) subjecting a DNA sequence encoding the parent (_(α)-amylaseto random mutagenesis,

[0123] (b) expressing the mutated DNA sequence obtained in step (a) in ahost cell, and

[0124] (c) screening for host cells expressing an _(α)-amylase variantwhich has an altered property (i.e. thermal stability) relative to theparent _(α)-amylase.

[0125] Step (a) of the above method of the invention is preferablyperformed using doped primers.

[0126] For instance, the random mutagenesis may be performed by use of asuitable physical or chemical mutagenizing agent, by use of a suitableoligonucleotide, or by subjecting the DNA sequence to PCR generatedmutagenesis. Furthermore, the random mutagenesis may be performed by useof any combination of these mutagenizing agents. The mutagenizing agentmay, e.g., be one which induces transitions, transversions, inversions,scrambling, deletions, and/or insertions.

[0127] Examples of a physical or chemical mutagenizing agent suitablefor the present purpose include ultraviolet (UV) ir-radiation,hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodiumbisulphite, formic acid, and nucleotide analogues. When such agents areused, the mutagenesis is typically performed by incubating the DNAsequence encoding the parent enzyme to be mutagenized in the presence ofthe mutagenizing agent of choice under suitable conditions for themutagenesis to take place, and selecting for mutated DNA having thedesired properties.

[0128] When the mutagenesis is performed by the use of anoligonucleotide, the oligonucleotide may be doped or spiked with thethree non-parent nucleotides during the synthesis of the oligonucleotideat the positions, which are to be changed. The doping or spiking may bedone so that codons for unwanted amino acids are avoided. The doped orspiked oligonucleotide can be incorporated into the DNA encoding thealpha-amylase enzyme by any published technique, using, e.g., PCR, LCRor any DNA polymerase and ligase as deemed appropriate.

[0129] Preferably, the doping is carried out using “constant randomdoping”, in which the percentage of wild-type and mutation in eachposition is predefined. Furthermore, the doping may be directed toward apreference for the introduction of certain nucleotides, and thereby apreference for the introduction of one or more specific amino acidresidues. The doping may be made, e.g., so as to allow for theintroduction of 90% wild type and 10% mutations in each position. Anadditional consideration in the choice of a doping scheme is based ongenetic as well as protein-structural constraints. The doping scheme maybe made by using the DOPE program, which, inter alia, ensures thatintroduction of stop codons is avoided.

[0130] When PCR-generated mutagenesis is used, either a chemicallytreated or non-treated gene encoding a parent alpha-amylase is subjectedto PCR under conditions that increase the mis-incorporation ofnucleotides (Deshler 1992; Leung et al., Technique, Vol.1, 1989, pp.11-15).

[0131] A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet.,133, 1974, pp. 179-191), S. cereviseae or any other microbial organismmay be used for the random mutagenesis of the DNA encoding thealpha-amylase by, e.g., transforming a plasmid containing the parentglycosylase into the mutator strain, growing the mutator strain with theplasmid and isolating the mutated plasmid from the mutator strain. Themutated plasmid may be subsequently transformed into the expressionorganism.

[0132] The DNA sequence to be mutagenized may be conveniently present ina genomic or cDNA library prepared from an organism expressing theparent alpha-amylase. Alternatively, the DNA sequence may be present ona suitable vector such as a plasmid or a bacteriophage, which as suchmay be incubated with or otherwise exposed to the mutagenising agent.The DNA to be mutagenized may also be present in a host cell either bybeing integrated in the genome of said cell or by being present on avector harboured in the cell. Finally, the DNA to be mutagenized may bein isolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence.

[0133] In some cases it may be convenient to amplify the mutated DNAsequence prior to performing the expression step b) or the screeningstep c). Such amplification may be performed in accordance with methodsknown in the art, the presently preferred method being PCR-generatedamplification using oligonucleotide primers prepared on the basis of theDNA or amino acid sequence of the parent enzyme.

[0134] Subsequent to the incubation with or exposure to the mutagenisingagent, the mutated DNA is expressed by culturing a suitable host cellcarrying the DNA sequence under conditions allowing expression to takeplace. The host cell used for this purpose may be one which has beentransformed with the mutated DNA sequence, optionally present on avector, or one which was carried the DNA sequence encoding the parentenzyme during the mutagenesis treatment. Examples of suitable host cellsare the following: gram positive bacteria such as Bacillus subtilis,Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermcphilus, Bacillus alkalophilus, Bacillus amryloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, Streptomyces lividans orStreptomyces murinus; and gram-negative bacteria such as E. coli.

[0135] The mutated DNA sequence may further comprise a DNA sequenceencoding functions permitting expression of the mutated DNA sequence.

Localized Random Mutagenesis

[0136] The random mutagenesis may be advantageously localized to a partof the parent alpha-amylase in question. This may, e.g., be advantageouswhen certain regions of the enzyme have been identified to be ofparticular importance for a given property of the enzyme, and whenmodified are expected to result in a variant having improved properties.Such regions may normally be identified when the tertiary structure ofthe parent enzyme has been elucidated and related to the function of theenzyme.

[0137] The localized, or region-specific, random mutagenesis isconveniently performed by use of PCR generated mutagenesis techniques asdescribed above or any other suitable technique known in the art.Alternatively, the DNA sequence encoding the part of the DNA sequence tobe modified may be isolated, e.g., by insertion into a suitable vector,and said part may be subsequently subjected to mutagenesis by use of anyof the mutagenesis methods discussed above.

Alternative Methods of Providing Alpha-Amylase Variants

[0138] Alternative methods for providing variants of the inventioninclude gene-shuffling method known in the art including the methods,e.g., described in WO 95/22625 (from Affymax Technologies N.V.) and WO96/00343 (from Novozymes A/S).

Expression of Alpha-Amylase Variants

[0139] According to the invention, a DNA sequence encoding the variantproduced by methods described above, or by any alternative methods knownin the art, can be expressed, in enzyme form, using an expression vectorwhich typically includes control sequences encoding a promoter,operator, ribosome binding site, translation initiation signal, and,optionally, a repressor gene or various activator genes.

[0140] The recombinant expression vector carrying the DNA sequenceencoding an alpha-amylase variant of the invention may be any vector,which may conveniently be subjected to recombinant DNA procedures, andthe choice of vector will often depend on the host cell into which it isto be introduced. Thus, the vector may be an autonomously replicatingvector, i.e., a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, a bacteriophage or an extrachromosomal element, minichromosomeor an artificial chromosome. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

[0141] In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence, whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA sequence encoding an alpha-amylase variant ofthe invention, especially in a bacterial host, are the promoter of thelac operon of E.coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis alpha-amylasegene (amyL), the promoters of the Bacillus stearothermophilus maltogenicamylase gene (amyM), the promoters of the Bacillus amylolique-faciensalpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA andxylB genes etc. For transcription in a fungal host, examples of usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralalpha-amylase, A. niger acid stable alpha-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase or A. nidulans acetamidase.

[0142] The expression vector of the invention may also comprise asuitable transcription terminator and, in eukaryotes, polyadenylationsequences operably connected to the DNA sequence encoding thealpha-amylase variant of the invention. Termination and polyadenylationsequences may suitably be derived from the same sources as the promoter.

[0143] The vector may further comprise a DNA sequence enabling thevector to replicate in the host cell in question. Examples of suchsequences are the origins of replication of plasmids pUC19, pACYC177,pUB110, pE194, pAMB1 and pIJ702.

[0144] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argE, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g., as described in WO 91/17243.

[0145] While intracellular expression may be advantageous in somerespects, e.g., when using certain bacteria as host cells, it isgenerally preferred that the expression is extracellular. In general,the Bacillus _(α)-amylases mentioned herein comprise a pre-regionpermitting secretion of the expressed protease into the culture medium.If desirable, this preregion may be replaced by a different preregion orsignal sequence, conveniently accomplished by substitution of the DNAsequences encoding the respective preregions.

[0146] The procedures used to ligate the DNA construct of the inventionencoding an alpha-amylase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989).

[0147] The cell of the invention, either comprising a DNA construct oran expression vector of the invention as defined above, isadvantageously used as a host cell in the recombinant production of analpha-amylase variant of the invention. The cell may be transformed withthe DNA construct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g., by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

[0148] The host cell of the invention may be a cell of a higher organismsuch as a mammal or an insect, but is preferably a microbial cell, e.g.,a bacterial or a fungal (including yeast) cell.

[0149] Examples of suitable bacteria are Gram-positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalo-philus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gramnegative bacteria such asE.coli. The transformation of the bacteria may, for instance, beeffected by protoplast transformation or by using competent cells in amanner known per se.

[0150] The yeast organism may favorably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. Thefilamentous fungus may advantageously belong to a species ofAspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cellsmay be transformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known per se. A suitable procedure for transformationof Aspergillus host cells is described in EP 238 023.

[0151] In a yet further aspect, the present invention relates to amethod of producing an alpha-amylase variant of the invention, whichmethod comprises cultivating a host cell as described above underconditions conducive to the production of the variant and recovering thevariant from the cells and/or culture medium.

[0152] The medium used to cultivate the cells may be any conventionalmedium suitable for growing the host cell in question and obtainingexpression of the alpha-amylase variant of the invention. Suitable mediaare available from commercial suppliers or may be prepared according topublished recipes (e.g., as described in catalogues of the American TypeCulture Collection).

[0153] The alpha-amylase variant secreted from the host cells mayconveniently be recovered from the culture medium by well-knownprocedures, including separating the cells from the medium bycentrifugation or filtration, and precipitating proteinaceous componentsof the medium by means of a salt such as ammonium sulphate, followed bythe use of chromatographic procedures such as ion exchangechromatography, affinity chromatography, or the like.

Industrial Applications

[0154] The alpha-amylase variants of this invention possess valuableproperties allowing for a variety of industrial applications. Inparticular, enzyme variants of the invention are applicable as acomponent in washing, dishwashing and hard surface cleaning detergentcompositions. Variant of the invention with altered properties may beused for starch processes, in particular starch conversion, especiallyliquefaction of starch (see, e.g., US 25 3,912,590, EP patentpublications Nos. 252 730 and 63 909, and WO 99/19467.

[0155] Further, variants of the invention are also particularly usefulin the production of sweeteners and ethanol from starch, and/or fortextile desizing.

Detergent Compositions

[0156] As mentioned above, variants of the invention may suitably beincorporated in detergent compositions. Reference is made, for example,to WO 96/23874 and WO 97/07202 for further details concerning relevantingredients of detergent compositions (such as laundry or dishwashingdetergents), appropriate methods of formulating the variants in suchdetergent compositions, and for examples of relevant types of detergentcompositions.

[0157] Detergent compositions comprising a variant of the invention mayadditionally comprise one or more other enzymes, such as a lipase,cutinase, protease, cellulase, peroxidase or laccase, and/or anotheralpha-amylase.

[0158] Alpha-amylase variants of the invention may be incorporated indetergents at conventionally employed concentrations. It is at presentcontemplated that a variant of the invention may be incorporated in anamount corresponding to 0.00001-10 mg (calculated as pure, active enzymeprotein) of alpha-amylase per liter of wash/dishwash liquor usingconventional dosing levels of detergent.

Materials & Methods Enzymes

[0159] TERMAMYL®: B. licheniformis alpha-amylase shown in SEQ ID NO: 8.NOVAMYL®: B. stearotherrmophilus C599 maltogenic amylase disclosed in EPpatent no. 120,693 (available from Novo Nordisk)

[0160] Bacillus subtilis SHA273: see WO 95/10603

Plasmids

[0161] PJE1 contains the gene encoding a variant of SP722 alpha-amylase(SEQ ID NO: 4): viz. deletion of 6 nucleotides corresponding to aminoacids D183-G184 in the mature protein. Transcription of the JEl gene isdirected from the amyL promoter. The plasmid further more contains theorigin of replication and cat-gene conferring resistance towardskanamycin obtained from plasmid pUB110 (Gryczan, TJ et al. (1978), J.Bact. 134:318-329).

Methods Construction of Library Vector PDorK101

[0162] The E. colil/Bacillus shuttle vector pDorK101 (described below)can be used to introduce mutations without expression of alpha-amylasein E. coli and then be modified in such way that the alpha-amylase isactive in Bacillus. The vector was constructed as follows: The JE1encoding gene (SP722 with the deletion of D183-G184) was inactivated inpJE1 by gene interruption in the PstI site in the 5′ coding region ofthe SEQ ID NO: 4: SP722 by a 1.2 kb fragment containing an E. coliorigin of replication. This fragment was PCR amplified from the pUC19(GenBank Accession #:X02514) using the forward primer:5′-gacctgcagtcaggcaacta-3′ (SEQ ID NO: 14) and the reverse primer:5′-tagagtcgacctgcaggcat-3′ (SEQ ID NO: 15). The PCR amplicon and thepJE1 vector were digested with PstI at 37° C. for 2 hours. The pJE1vector fragment and the PCR fragment were ligated at room temperature.for 1 hour and transformed in E. coli by electrotransformation. Theresulting vector is designated pDorK101.

Fermentation and Purification of Alpha-Amylase Variants

[0163] Fermentation and purification may be performed by methods wellknown in the art.

Procedure for Determination of 1,6-Activity

[0164] The enzyme solutions (of a chosen activity, e.g., 10-100 NU) arediluted with D₂O and freeze-dried. The samples are re-dissolved in D₂O(0.5 mL) and freeze-dried. Samples containing 25 mg of substrate in D₂O(0.5 mL) are freeze-dried before re-dissolved (D₂O, 0.5 mL) andfreeze-dried. Finally the enzymes are dissolved in D₂O (1 mL) and addedto each sample of substrate. The solutions are transferred to NMR tubesand incubated at 60° C. ¹H NMR spectra are recorded currently at 60° C.on a Varian Mercury 400 MHz instrument (5 mm inverse probe head, 32scan).

Preparation of NOVAMYL® and TERMAMYL® Limit Dextrins

[0165] The NOVAMYL® limit dextrin is prepared from waxy maize starch(100% amylopectin) by gelatinization at 80° C. and treatment withNOVAMYL® for 4 hours at 60° C. The enzyme is inactivated at 100° C. (10minutes) before smaller dextrins are removed by ultrafiltration. Thedextrin is then be obtained as white fibers after freeze-drying.

[0166] The Termamyl limit dextrin is prepared by a similar procedure;gelatinisation at 100° C., incubation for 2 hours at 90° C.,inactivation, ultrafiltration and freeze-drying. See also Mottawia etal, Carbohydr. Res. 277 (1995), 109-123 (purification and beta-amylasetreatment excluded in the present procedure).

Assays for Alpha-Amylase Activity

[0167] 1. Phadebas Assay

[0168] Alpha-amylase activity is determined by a method employingPHADEBAS® tablets as substrate. Phadebas tablets (PHADEBAS® AmylaseTest, supplied by Pharmacia Diagnostic) contain a cross-linked insolubleblue-colored starch polymer, which has been mixed with bovine serumalbumin and a buffer substance and tabletted.

[0169] For every single measurement one tablet is suspended in a tubecontaining 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, pH adjusted to thevalue of interest with NaOH). The test is performed in a water bath atthe temperature of interest. The alpha-amylase to be tested is dilutedin ×ml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylasesolution is added to the 5 ml 50 mM Britton-Robinson buffer. The starchis hydrolyzed by the alpha-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the alpha-amylaseactivity.

[0170] It is important that the measured 620 nm absorbance after 10 or15 minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units at 620 nm. In this absorbance range there is linearitybetween activity and absorbance (Lambert-Beer law) The dilution of theenzyme must therefore be adjusted to fit this criterion. Under aspecified set of conditions (temp., pH, reaction time, bufferconditions) 1 mg of a given alpha-amylase will hydrolyze a certainamount of substrate and a blue colour will be produced. The colourintensity is measured at 620 nm. The measured absorbance is directlyproportional to the specific activity (activity/mg of pure alpha-amylaseprotein) of the alpha-amylase in question under the given set ofconditions.

[0171] 2. Alternative Method

[0172] Alpha-amylase activity is determined by a method employing thePNP-G₇ substrate. PNP-G₇ which is a abbreviation forp-nitrophenyl-alpha,D-maltoheptaoside is a blocked oligosaccharide whichcan be cleaved by an endo-amylase. Following the cleavage, thealpha-Glucosidase included in the kit digest the substrate to liberate afree PNP molecule which has a yellow colour and thus can be measured byvisible spectophometry at _(γ)=405 nm. (400-420 nm.). Kits containingPNP-G₇ substrate and alpha-Glucosidase is manufactured byBoehringer-Mannheim (cat. No. 1054635).

[0173] To prepare the substrate one bottle of substrate (BM 1442309) isadded to 5 ml buffer (BM1442309). To prepare the _(α)-Glucosidase onebottle of alpha-Glucosidase (BM 1462309) is added to 45 ml buffer(BM1442309). The working solution is made by mixing 5 mlalpha-Glucosidase solution with 0.5 ml substrate.

[0174] The assay is performed by transforming 20 micro 1 enzyme solutionto a 96 well microtitre plate and incubating at 25° C. 200 micro 1working solution, 25° C. is added. The solution is mixed andpre-incubated 1 minute and absorption is measured every 15 second over 3minutes at OD 405 nm.

[0175] The slope of the time dependent absorption-curve is directlyproportional to the specific activity (activity per mg enzyme) of thealpha-amylase in question under the given set of conditions.

General Method for Random Mutagenesis by use of the DOPE Program

[0176] The random mutagenesis may be carried out as follows:

[0177] 1. Select regions of interest for modification in the parentenzyme

[0178] 2. Decide on mutation sites and non-mutated sites in the selectedregion

[0179] 3. Decide on which kind of mutations should be carried out, e.g.with respect to the desired stability and/or performance of the variantto be constructed

[0180] 4. Select structurally reasonable mutations.

[0181] 5. Adjust the residues selected by step 3 with regard to step 4.

[0182] 6. Analyze by use of a suitable dope algorithm the nucleotidedistribution.

[0183] 7. If necessary, adjust the wanted residues to genetic coderealism (e.g., taking into account constraints resulting from thegenetic code (e.g. in order to avoid introduction of stop codons))(theskilled person will be aware that some codon combinations cannot be usedin practice and will need to be adapted)

[0184] 8. Make primers

[0185] 9. Perform random mutagenesis by use of the primers

[0186] 10. Select resulting alpha-amylase variants by screening for thedesired improved properties.

[0187] Suitable dope algorithms for use in step 6 are well known in theart. One algorithm is described by Tomandl, D. et al., Journal ofComputer-Aided Molecular Design, 11 (1997), pp. 29-38). Anotheralgorithm, DOPE, is described in the following:

The Dope Program

[0188] The “DOPE” program is a computer algorithm useful to optimize thenucleotide composition of a codon triplet in such a way that it encodesan amino acid distribution, which resembles most the wanted amino aciddistribution. In order to assess which of the possible distributions isthe most similar to the wanted amino acid distribution, a scoringfunction is needed. In the “Dope” program the following function wasfound to be suited:${s \equiv {\prod\limits_{i = 1}^{N}\left( {\frac{x_{i}^{y_{i}}}{y_{i}^{y_{i}}}\frac{\left( {1 - x_{i}} \right)^{1 - y_{i}}}{\left( {1 - y_{i}} \right)^{1 - y_{i}}}} \right)^{w_{i}}}},$

[0189] where the x₁'s are the obtained amounts of amino acids and groupsof amino acids as calculated by the program, y₁'s are the wanted amountsof amino acids and groups of amino acids as defined by the user of theprogram (e.g. specify which of the 20 amino acids or stop codons arewanted to be introduced, e.g., with a certain percentage (e.g. 90% Ala,3% Ile, 7% Val), and w₁'s are assigned weight factors as defined by theuser of the program (e.g., depending on the importance of having aspecific amino acid residue inserted into the position in question). Nis 21 plus the number of amino acid groups as defined by the user of theprogram. For purposes of this function 0⁰ is defined as being 1.

[0190] A Monte-Carlo algorithm (one example being the one described byValleau, J. P. & Whittington, S. G. (1977) A guide to Mont Carlo forstatistical mechanics: 1 Highways. In “Stastistical Mechanics, Part A”Equlibrium Techniqeues ed. B. J. Berne, New York: Plenum) is used forfinding the maximum value of this function. In each iteration thefollowing steps are performed:

[0191] 1.A new random nucleotide composition is chosen for each base,where the absolute difference between the current and the newcomposition is smaller than or equal to d for each of the fournucleotides G,A,T,C in all three positions of the codon (see below fordefinition of d).

[0192] 2.The scores of the new composition and the current compositionare compared by the use of the function s as described above. If the newscore is higher or equal to the score of the current composition, thenew composition is kept and the current composition is changed to thenew one. If the new score is smaller, the probability of keeping the newcomposition is exp(1000(new_score-current_score)).

[0193] A cycle normally consists of 1000 iterations as described abovein which d is decreasing linearly from 1 to 0. One hundred or morecycles are performed in an optimization process. The nucleotidecomposition resulting in the highest score is finally presented.

EXAMPLES Example 1 Construction of Variants of the Invention andDetermination of Altered 1,6-Activity

[0194] The following variants are constructed as described in EXAMPLE 1of WO 00/29560 (from Novozymes A/S) in the Bacillus licheniformisalpha-amylase shown in SEQID NO: 8:

[0195] W263GA,S,T,V;

[0196] N265G,A,S,T,V;

[0197] V286F,W,Y,G,A,S;

[0198] Y290A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,V;

[0199] L335G,A,S,T,V; and

[0200] K234N,Q.

[0201] The altered 1,6-activity is determined in comparison to the paredas described in the Materials & Methods″ section above under ″Procedurefor determination of 1,6-activity

.

1 15 1 1455 DNA Bacillus sp. CDS (1)..(1455) SP690 1 cat cat aat gga acaaat ggt act atg atg caa tat ttc gaa tgg tat 48 His His Asn Gly Thr AsnGly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 ttg cca aat gac gggaat cat tgg aac agg ttg agg gat gac gca gct 96 Leu Pro Asn Asp Gly AsnHis Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 aac tta aag agt aaa gggata aca gct gta tgg atc cca cct gca tgg 144 Asn Leu Lys Ser Lys Gly IleThr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 aag ggg act tcc cag aat gatgta ggt tat gga gcc tat gat tta tat 192 Lys Gly Thr Ser Gln Asn Asp ValGly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 gat ctt gga gag ttt aac cag aagggg acg gtt cgt aca aaa tat gga 240 Asp Leu Gly Glu Phe Asn Gln Lys GlyThr Val Arg Thr Lys Tyr Gly 65 70 75 80 aca cgc aac cag cta cag gct gcggtg acc tct tta aaa aat aac ggc 288 Thr Arg Asn Gln Leu Gln Ala Ala ValThr Ser Leu Lys Asn Asn Gly 85 90 95 att cag gta tat ggt gat gtc gtc atgaat cat aaa ggt gga gca gat 336 Ile Gln Val Tyr Gly Asp Val Val Met AsnHis Lys Gly Gly Ala Asp 100 105 110 ggt acg gaa att gta aat gcg gta gaagtg aat cgg agc aac cga aac 384 Gly Thr Glu Ile Val Asn Ala Val Glu ValAsn Arg Ser Asn Arg Asn 115 120 125 cag gaa acc tca gga gag tat gca atagaa gcg tgg aca aag ttt gat 432 Gln Glu Thr Ser Gly Glu Tyr Ala Ile GluAla Trp Thr Lys Phe Asp 130 135 140 ttt cct gga aga gga aat aac cat tccagc ttt aag tgg cgc tgg tat 480 Phe Pro Gly Arg Gly Asn Asn His Ser SerPhe Lys Trp Arg Trp Tyr 145 150 155 160 cat ttt gat ggg aca gat tgg gatcag tca cgc cag ctt caa aac aaa 528 His Phe Asp Gly Thr Asp Trp Asp GlnSer Arg Gln Leu Gln Asn Lys 165 170 175 ata tat aaa ttc agg gga aca ggcaag gcc tgg gac tgg gaa gtc gat 576 Ile Tyr Lys Phe Arg Gly Thr Gly LysAla Trp Asp Trp Glu Val Asp 180 185 190 aca gag aat ggc aac tat gac tatctt atg tat gca gac gtg gat atg 624 Thr Glu Asn Gly Asn Tyr Asp Tyr LeuMet Tyr Ala Asp Val Asp Met 195 200 205 gat cac cca gaa gta ata cat gaactt aga aac tgg gga gtg tgg tat 672 Asp His Pro Glu Val Ile His Glu LeuArg Asn Trp Gly Val Trp Tyr 210 215 220 acg aat aca ctg aac ctt gat ggattt aga ata gat gca gtg aaa cat 720 Thr Asn Thr Leu Asn Leu Asp Gly PheArg Ile Asp Ala Val Lys His 225 230 235 240 ata aaa tat agc ttt acg agagat tgg ctt aca cat gtg cgt aac acc 768 Ile Lys Tyr Ser Phe Thr Arg AspTrp Leu Thr His Val Arg Asn Thr 245 250 255 aca ggt aaa cca atg ttt gcagtg gct gag ttt tgg aaa aat gac ctt 816 Thr Gly Lys Pro Met Phe Ala ValAla Glu Phe Trp Lys Asn Asp Leu 260 265 270 ggt gca att gaa aac tat ttgaat aaa aca agt tgg aat cac tcg gtg 864 Gly Ala Ile Glu Asn Tyr Leu AsnLys Thr Ser Trp Asn His Ser Val 275 280 285 ttt gat gtt cct ctc cac tataat ttg tac aat gca tct aat agc ggt 912 Phe Asp Val Pro Leu His Tyr AsnLeu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 ggt tat tat gat atg aga aatatt tta aat ggt tct gtg gtg caa aaa 960 Gly Tyr Tyr Asp Met Arg Asn IleLeu Asn Gly Ser Val Val Gln Lys 305 310 315 320 cat cca aca cat gcc gttact ttt gtt gat aac cat gat tct cag ccc 1008 His Pro Thr His Ala Val ThrPhe Val Asp Asn His Asp Ser Gln Pro 325 330 335 ggg gaa gca ttg gaa tccttt gtt caa caa tgg ttt aaa cca ctt gca 1056 Gly Glu Ala Leu Glu Ser PheVal Gln Gln Trp Phe Lys Pro Leu Ala 340 345 350 tat gca ttg gtt ctg acaagg gaa caa ggt tat cct tcc gta ttt tat 1104 Tyr Ala Leu Val Leu Thr ArgGlu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 ggg gat tac tac ggt atccca acc cat ggt gtt ccg gct atg aaa tct 1152 Gly Asp Tyr Tyr Gly Ile ProThr His Gly Val Pro Ala Met Lys Ser 370 375 380 aaa ata gac cct ctt ctgcag gca cgt caa act ttt gcc tat ggt acg 1200 Lys Ile Asp Pro Leu Leu GlnAla Arg Gln Thr Phe Ala Tyr Gly Thr 385 390 395 400 cag cat gat tac tttgat cat cat gat att atc ggt tgg aca aga gag 1248 Gln His Asp Tyr Phe AspHis His Asp Ile Ile Gly Trp Thr Arg Glu 405 410 415 gga aat agc tcc catcca aat tca ggc ctt gcc acc att atg tca gat 1296 Gly Asn Ser Ser His ProAsn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 ggt cca ggt ggt aacaaa tgg atg tat gtg ggg aaa aat aaa gcg gga 1344 Gly Pro Gly Gly Asn LysTrp Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445 caa gtt tgg aga gatatt acc gga aat agg aca ggc acc gtc aca att 1392 Gln Val Trp Arg Asp IleThr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 aat gca gac gga tggggt aat ttc tct gtt aat gga ggg tcc gtt tcg 1440 Asn Ala Asp Gly Trp GlyAsn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 gtt tgg gtg aagcaa 1455 Val Trp Val Lys Gln 485 2 485 PRT Bacillus sp. 2 His His AsnGly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu ProAsn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 Asn LeuLys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys GlyThr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp LeuGly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 ThrArg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 IleGln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120125 Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp 130135 140 Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr145 150 155 160 His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu GlnAsn Lys 165 170 175 Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp TrpGlu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr AlaAsp Val Asp Met 195 200 205 Asp His Pro Glu Val Ile His Glu Leu Arg AsnTrp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe ArgIle Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg AspTrp Leu Thr His Val Arg Asn Thr 245 250 255 Thr Gly Lys Pro Met Phe AlaVal Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn TyrLeu Asn Lys Thr Ser Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro LeuHis Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Tyr Tyr AspMet Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys 305 310 315 320 His ProThr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 GlyGlu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala 340 345 350Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370375 380 Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr385 390 395 400 Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp ThrArg Glu 405 410 415 Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr IleMet Ser Asp 420 425 430 Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly LysAsn Lys Ala Gly 435 440 445 Gln Val Trp Arg Asp Ile Thr Gly Asn Arg ThrGly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser ValAsn Gly Gly Ser Val Ser 465 470 475 480 Val Trp Val Lys Gln 485 3 1455DNA Bacillus sp. CDS (1)..(1455) SP722 3 cat cat aat ggg aca aat ggg acgatg atg caa tac ttt gaa tgg cac 48 His His Asn Gly Thr Asn Gly Thr MetMet Gln Tyr Phe Glu Trp His 1 5 10 15 ttg cct aat gat ggg aat cac tggaat aga tta aga gat gat gct agt 96 Leu Pro Asn Asp Gly Asn His Trp AsnArg Leu Arg Asp Asp Ala Ser 20 25 30 aat cta aga aat aga ggt ata acc gctatt tgg att ccg cct gcc tgg 144 Asn Leu Arg Asn Arg Gly Ile Thr Ala IleTrp Ile Pro Pro Ala Trp 35 40 45 aaa ggg act tcg caa aat gat gtg ggg tatgga gcc tat gat ctt tat 192 Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr GlyAla Tyr Asp Leu Tyr 50 55 60 gat tta ggg gaa ttt aat caa aag ggg acg gttcgt act aag tat ggg 240 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val ArgThr Lys Tyr Gly 65 70 75 80 aca cgt agt caa ttg gag tct gcc atc cat gcttta aag aat aat ggc 288 Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala LeuLys Asn Asn Gly 85 90 95 gtt caa gtt tat ggg gat gta gtg atg aac cat aaagga gga gct gat 336 Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys GlyGly Ala Asp 100 105 110 gct aca gaa aac gtt ctt gct gtc gag gtg aat ccaaat aac cgg aat 384 Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro AsnAsn Arg Asn 115 120 125 caa gaa ata tct ggg gac tac aca att gag gct tggact aag ttt gat 432 Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp ThrLys Phe Asp 130 135 140 ttt cca ggg agg ggt aat aca tac tca gac ttt aaatgg cgt tgg tat 480 Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys TrpArg Trp Tyr 145 150 155 160 cat ttc gat ggt gta gat tgg gat caa tca cgacaa ttc caa aat cgt 528 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg GlnPhe Gln Asn Arg 165 170 175 atc tac aaa ttc cga ggt gat ggt aag gca tgggat tgg gaa gta gat 576 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp AspTrp Glu Val Asp 180 185 190 tcg gaa aat gga aat tat gat tat tta atg tatgca gat gta gat atg 624 Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr AlaAsp Val Asp Met 195 200 205 gat cat ccg gag gta gta aat gag ctt aga agatgg gga gaa tgg tat 672 Asp His Pro Glu Val Val Asn Glu Leu Arg Arg TrpGly Glu Trp Tyr 210 215 220 aca aat aca tta aat ctt gat gga ttt agg atcgat gcg gtg aag cat 720 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile AspAla Val Lys His 225 230 235 240 att aaa tat agc ttt aca cgt gat tgg ttgacc cat gta aga aac gca 768 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu ThrHis Val Arg Asn Ala 245 250 255 acg gga aaa gaa atg ttt gct gtt gct gaattt tgg aaa aat gat tta 816 Thr Gly Lys Glu Met Phe Ala Val Ala Glu PheTrp Lys Asn Asp Leu 260 265 270 ggt gcc ttg gag aac tat tta aat aaa acaaac tgg aat cat tct gtc 864 Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr AsnTrp Asn His Ser Val 275 280 285 ttt gat gtc ccc ctt cat tat aat ctt tataac gcg tca aat agt gga 912 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr AsnAla Ser Asn Ser Gly 290 295 300 ggc aac tat gac atg gca aaa ctt ctt aatgga acg gtt gtt caa aag 960 Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn GlyThr Val Val Gln Lys 305 310 315 320 cat cca atg cat gcc gta act ttt gtggat aat cac gat tct caa cct 1008 His Pro Met His Ala Val Thr Phe Val AspAsn His Asp Ser Gln Pro 325 330 335 ggg gaa tca tta gaa tca ttt gta caagaa tgg ttt aag cca ctt gct 1056 Gly Glu Ser Leu Glu Ser Phe Val Gln GluTrp Phe Lys Pro Leu Ala 340 345 350 tat gcg ctt att tta aca aga gaa caaggc tat ccc tct gtc ttc tat 1104 Tyr Ala Leu Ile Leu Thr Arg Glu Gln GlyTyr Pro Ser Val Phe Tyr 355 360 365 ggt gac tac tat gga att cca aca catagt gtc cca gca atg aaa gcc 1152 Gly Asp Tyr Tyr Gly Ile Pro Thr His SerVal Pro Ala Met Lys Ala 370 375 380 aag att gat cca atc tta gag gcg cgtcaa aat ttt gca tat gga aca 1200 Lys Ile Asp Pro Ile Leu Glu Ala Arg GlnAsn Phe Ala Tyr Gly Thr 385 390 395 400 caa cat gat tat ttt gac cat cataat ata atc gga tgg aca cgt gaa 1248 Gln His Asp Tyr Phe Asp His His AsnIle Ile Gly Trp Thr Arg Glu 405 410 415 gga aat acc acg cat ccc aat tcagga ctt gcg act atc atg tcg gat 1296 Gly Asn Thr Thr His Pro Asn Ser GlyLeu Ala Thr Ile Met Ser Asp 420 425 430 ggg cca ggg gga gag aaa tgg atgtac gta ggg caa aat aaa gca ggt 1344 Gly Pro Gly Gly Glu Lys Trp Met TyrVal Gly Gln Asn Lys Ala Gly 435 440 445 caa gtt tgg cat gac ata act ggaaat aaa cca gga aca gtt acg atc 1392 Gln Val Trp His Asp Ile Thr Gly AsnLys Pro Gly Thr Val Thr Ile 450 455 460 aat gca gat gga tgg gct aat ttttca gta aat gga gga tct gtt tcc 1440 Asn Ala Asp Gly Trp Ala Asn Phe SerVal Asn Gly Gly Ser Val Ser 465 470 475 480 att tgg gtg aaa cga 1455 IleTrp Val Lys Arg 485 4 485 PRT Bacillus sp. 4 His His Asn Gly Thr Asn GlyThr Met Met Gln Tyr Phe Glu Trp His 1 5 10 15 Leu Pro Asn Asp Gly AsnHis Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30 Asn Leu Arg Asn Arg GlyIle Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln AsnAsp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe AsnGln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Ser Gln LeuGlu Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85 90 95 Val Gln Val Tyr GlyAsp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu AsnVal Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu IleSer Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe ProGly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr 145 150 155 160His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg 165 170175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp 180185 190 Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu TrpTyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala ValLys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr HisVal Arg Asn Ala 245 250 255 Thr Gly Lys Glu Met Phe Ala Val Ala Glu PheTrp Lys Asn Asp Leu 260 265 270 Gly Ala Leu Glu Asn Tyr Leu Asn Lys ThrAsn Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn LeuTyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Asn Tyr Asp Met Ala Lys LeuLeu Asn Gly Thr Val Val Gln Lys 305 310 315 320 His Pro Met His Ala ValThr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Gly Glu Ser Leu GluSer Phe Val Gln Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu IleLeu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp TyrTyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala 370 375 380 Lys IleAsp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr 385 390 395 400Gln His Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410415 Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420425 430 Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly435 440 445 Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val ThrIle 450 455 460 Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly SerVal Ser 465 470 475 480 Ile Trp Val Lys Arg 485 5 1548 DNA Bacillusstearothermophilus CDS (1)..(1548) BSG 5 gcc gca ccg ttt aac ggc acc atgatg cag tat ttt gaa tgg tac ttg 48 Ala Ala Pro Phe Asn Gly Thr Met MetGln Tyr Phe Glu Trp Tyr Leu 1 5 10 15 ccg gat gat ggc acg tta tgg accaaa gtg gcc aat gaa gcc aac aac 96 Pro Asp Asp Gly Thr Leu Trp Thr LysVal Ala Asn Glu Ala Asn Asn 20 25 30 tta tcc agc ctt ggc atc acc gct ctttgg ctg ccg ccc gct tac aaa 144 Leu Ser Ser Leu Gly Ile Thr Ala Leu TrpLeu Pro Pro Ala Tyr Lys 35 40 45 gga aca agc cgc agc gac gta ggg tac ggagta tac gac ttg tat gac 192 Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly ValTyr Asp Leu Tyr Asp 50 55 60 ctc ggc gaa ttc aat caa aaa ggg acc gtc cgcaca aaa tac gga aca 240 Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg ThrLys Tyr Gly Thr 65 70 75 80 aaa gct caa tat ctt caa gcc att caa gcc gcccac gcc gct gga atg 288 Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala HisAla Ala Gly Met 85 90 95 caa gtg tac gcc gat gtc gtg ttc gac cat aaa ggcggc gct gac ggc 336 Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly GlyAla Asp Gly 100 105 110 acg gaa tgg gtg gac gcc gtc gaa gtc aat ccg tccgac cgc aac caa 384 Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser AspArg Asn Gln 115 120 125 gaa atc tcg ggc acc tat caa atc caa gca tgg acgaaa ttt gat ttt 432 Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr LysPhe Asp Phe 130 135 140 ccc ggg cgg ggc aac acc tac tcc agc ttt aag tggcgc tgg tac cat 480 Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp ArgTrp Tyr His 145 150 155 160 ttt gac ggc gtt gat tgg gac gaa agc cga aaattg agc cgc att tac 528 Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys LeuSer Arg Ile Tyr 165 170 175 aaa ttc cgc ggc atc ggc aaa gcg tgg gat tgggaa gta gac acg gaa 576 Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp GluVal Asp Thr Glu 180 185 190 aac gga aac tat gac tac tta atg tat gcc gacctt gat atg gat cat 624 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp LeuAsp Met Asp His 195 200 205 ccc gaa gtc gtg acc gag ctg aaa aac tgg gggaaa tgg tat gtc aac 672 Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly LysTrp Tyr Val Asn 210 215 220 aca acg aac att gat ggg ttc cgg ctt gat gccgtc aag cat att aag 720 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala ValLys His Ile Lys 225 230 235 240 ttc agt ttt ttt cct gat tgg ttg tcg tatgtg cgt tct cag act ggc 768 Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr ValArg Ser Gln Thr Gly 245 250 255 aag ccg cta ttt acc gtc ggg gaa tat tggagc tat gac atc aac aag 816 Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp SerTyr Asp Ile Asn Lys 260 265 270 ttg cac aat tac att acg aaa aca gac ggaacg atg tct ttg ttt gat 864 Leu His Asn Tyr Ile Thr Lys Thr Asp Gly ThrMet Ser Leu Phe Asp 275 280 285 gcc ccg tta cac aac aaa ttt tat acc gcttcc aaa tca ggg ggc gca 912 Ala Pro Leu His Asn Lys Phe Tyr Thr Ala SerLys Ser Gly Gly Ala 290 295 300 ttt gat atg cgc acg tta atg acc aat actctc atg aaa gat caa ccg 960 Phe Asp Met Arg Thr Leu Met Thr Asn Thr LeuMet Lys Asp Gln Pro 305 310 315 320 aca ttg gcc gtc acc ttc gtt gat aatcat gac acc gaa ccc ggc caa 1008 Thr Leu Ala Val Thr Phe Val Asp Asn HisAsp Thr Glu Pro Gly Gln 325 330 335 gcg ctg cag tca tgg gtc gac cca tggttc aaa ccg ttg gct tac gcc 1056 Ala Leu Gln Ser Trp Val Asp Pro Trp PheLys Pro Leu Ala Tyr Ala 340 345 350 ttt att cta act cgg cag gaa gga tacccg tgc gtc ttt tat ggt gac 1104 Phe Ile Leu Thr Arg Gln Glu Gly Tyr ProCys Val Phe Tyr Gly Asp 355 360 365 tat tat ggc att cca caa tat aac attcct tcg ctg aaa agc aaa atc 1152 Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile ProSer Leu Lys Ser Lys Ile 370 375 380 gat ccg ctc ctc atc gcg cgc agg gattat gct tac gga acg caa cat 1200 Asp Pro Leu Leu Ile Ala Arg Arg Asp TyrAla Tyr Gly Thr Gln His 385 390 395 400 gat tat ctt gat cac tcc gac atcatc ggg tgg aca agg gaa ggg ggc 1248 Asp Tyr Leu Asp His Ser Asp Ile IleGly Trp Thr Arg Glu Gly Gly 405 410 415 act gaa aaa cca gga tcc gga ctggcc gca ctg atc acc gat ggg ccg 1296 Thr Glu Lys Pro Gly Ser Gly Leu AlaAla Leu Ile Thr Asp Gly Pro 420 425 430 gga gga agc aaa tgg atg tac gttggc aaa caa cac gct gga aaa gtg 1344 Gly Gly Ser Lys Trp Met Tyr Val GlyLys Gln His Ala Gly Lys Val 435 440 445 ttc tat gac ctt acc ggc aac cggagt gac acc gtc acc atc aac agt 1392 Phe Tyr Asp Leu Thr Gly Asn Arg SerAsp Thr Val Thr Ile Asn Ser 450 455 460 gat gga tgg ggg gaa ttc aaa gtcaat ggc ggt tcg gtt tcg gtt tgg 1440 Asp Gly Trp Gly Glu Phe Lys Val AsnGly Gly Ser Val Ser Val Trp 465 470 475 480 gtt cct aga aaa acg acc gtttct acc atc gct cgg ccg atc aca acc 1488 Val Pro Arg Lys Thr Thr Val SerThr Ile Ala Arg Pro Ile Thr Thr 485 490 495 cga ccg tgg act ggt gaa ttcgtc cgt tgg acc gaa cca cgg ttg gtg 1536 Arg Pro Trp Thr Gly Glu Phe ValArg Trp Thr Glu Pro Arg Leu Val 500 505 510 gca tgg cct tga 1548 Ala TrpPro 515 6 515 PRT Bacillus stearothermophilus 6 Ala Ala Pro Phe Asn GlyThr Met Met Gln Tyr Phe Glu Trp Tyr Leu 1 5 10 15 Pro Asp Asp Gly ThrLeu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30 Leu Ser Ser Leu GlyIle Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45 Gly Thr Ser Arg SerAsp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55 60 Leu Gly Glu Phe AsnGln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr 65 70 75 80 Lys Ala Gln TyrLeu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met 85 90 95 Gln Val Tyr AlaAsp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly 100 105 110 Thr Glu TrpVal Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln 115 120 125 Glu IleSer Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe 130 135 140 ProGly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His 145 150 155160 Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165170 175 Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu180 185 190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met AspHis 195 200 205 Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp TyrVal Asn 210 215 220 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val LysHis Ile Lys 225 230 235 240 Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr ValArg Ser Gln Thr Gly 245 250 255 Lys Pro Leu Phe Thr Val Gly Glu Tyr TrpSer Tyr Asp Ile Asn Lys 260 265 270 Leu His Asn Tyr Ile Thr Lys Thr AspGly Thr Met Ser Leu Phe Asp 275 280 285 Ala Pro Leu His Asn Lys Phe TyrThr Ala Ser Lys Ser Gly Gly Ala 290 295 300 Phe Asp Met Arg Thr Leu MetThr Asn Thr Leu Met Lys Asp Gln Pro 305 310 315 320 Thr Leu Ala Val ThrPhe Val Asp Asn His Asp Thr Glu Pro Gly Gln 325 330 335 Ala Leu Gln SerTrp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Phe Ile LeuThr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp 355 360 365 Tyr TyrGly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile 370 375 380 AspPro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His 385 390 395400 Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly 405410 415 Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro420 425 430 Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly LysVal 435 440 445 Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr IleAsn Ser 450 455 460 Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser ValSer Val Trp 465 470 475 480 Val Pro Arg Lys Thr Thr Val Ser Thr Ile AlaArg Pro Ile Thr Thr 485 490 495 Arg Pro Trp Thr Gly Glu Phe Val Arg TrpThr Glu Pro Arg Leu Val 500 505 510 Ala Trp Pro 515 7 1920 DNA Bacilluslicheniformis CDS (421)..(1872) TERMAMYL 7 cggaagattg gaagtacaaaaataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60 gagacggaaa aatcgtcttaatgcacgata tttatgcaac gttcgcagat gctgctgaag 120 agattattaa aaagctgaaagcaaaaggct atcaattggt aactgtatct cagcttgaag 180 aagtgaagaa gcagagaggctattgaataa atgagtagaa gcgccatatc ggcgcttttc 240 ttttggaaga aaatatagggaaaatggtac ttgttaaaaa ttcggaatat ttatacaaca 300 tcatatgttt cacattgaaaggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360 cgattgctga cgctgttatttgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420 gca aat ctt aat ggg acgctg atg cag tat ttt gaa tgg tac atg ccc 468 Ala Asn Leu Asn Gly Thr LeuMet Gln Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 aat gac ggc caa cat tggagg cgt ttg caa aac gac tcg gca tat ttg 516 Asn Asp Gly Gln His Trp ArgArg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30 gct gaa cac ggt att act gccgtc tgg att ccc ccg gca tat aag gga 564 Ala Glu His Gly Ile Thr Ala ValTrp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 acg agc caa gcg gat gtg ggc tacggt gct tac gac ctt tat gat tta 612 Thr Ser Gln Ala Asp Val Gly Tyr GlyAla Tyr Asp Leu Tyr Asp Leu 50 55 60 ggg gag ttt cat caa aaa ggg acg gttcgg aca aag tac ggc aca aaa 660 Gly Glu Phe His Gln Lys Gly Thr Val ArgThr Lys Tyr Gly Thr Lys 65 70 75 80 gga gag ctg caa tct gcg atc aaa agtctt cat tcc cgc gac att aac 708 Gly Glu Leu Gln Ser Ala Ile Lys Ser LeuHis Ser Arg Asp Ile Asn 85 90 95 gtt tac ggg gat gtg gtc atc aac cac aaaggc ggc gct gat gcg acc 756 Val Tyr Gly Asp Val Val Ile Asn His Lys GlyGly Ala Asp Ala Thr 100 105 110 gaa gat gta acc gcg gtt gaa gtc gat cccgct gac cgc aac cgc gta 804 Glu Asp Val Thr Ala Val Glu Val Asp Pro AlaAsp Arg Asn Arg Val 115 120 125 att tca gga gaa cac cta att aaa gcc tggaca cat ttt cat ttt ccg 852 Ile Ser Gly Glu His Leu Ile Lys Ala Trp ThrHis Phe His Phe Pro 130 135 140 ggg cgc ggc agc aca tac agc gat ttt aaatgg cat tgg tac cat ttt 900 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys TrpHis Trp Tyr His Phe 145 150 155 160 gac gga acc gat tgg gac gag tcc cgaaag ctg aac cgc atc tat aag 948 Asp Gly Thr Asp Trp Asp Glu Ser Arg LysLeu Asn Arg Ile Tyr Lys 165 170 175 ttt caa gga aag gct tgg gat tgg gaagtt tcc aat gaa aac ggc aac 996 Phe Gln Gly Lys Ala Trp Asp Trp Glu ValSer Asn Glu Asn Gly Asn 180 185 190 tat gat tat ttg atg tat gcc gac atcgat tat gac cat cct gat gtc 1044 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile AspTyr Asp His Pro Asp Val 195 200 205 gca gca gaa att aag aga tgg ggc acttgg tat gcc aat gaa ctg caa 1092 Ala Ala Glu Ile Lys Arg Trp Gly Thr TrpTyr Ala Asn Glu Leu Gln 210 215 220 ttg gac ggt ttc cgt ctt gat gct gtcaaa cac att aaa ttt tct ttt 1140 Leu Asp Gly Phe Arg Leu Asp Ala Val LysHis Ile Lys Phe Ser Phe 225 230 235 240 ttg cgg gat tgg gtt aat cat gtcagg gaa aaa acg ggg aag gaa atg 1188 Leu Arg Asp Trp Val Asn His Val ArgGlu Lys Thr Gly Lys Glu Met 245 250 255 ttt acg gta gct gaa tat tgg cagaat gac ttg ggc gcg ctg gaa aac 1236 Phe Thr Val Ala Glu Tyr Trp Gln AsnAsp Leu Gly Ala Leu Glu Asn 260 265 270 tat ttg aac aaa aca aat ttt aatcat tca gtg ttt gac gtg ccg ctt 1284 Tyr Leu Asn Lys Thr Asn Phe Asn HisSer Val Phe Asp Val Pro Leu 275 280 285 cat tat cag ttc cat gct gca tcgaca cag gga ggc ggc tat gat atg 1332 His Tyr Gln Phe His Ala Ala Ser ThrGln Gly Gly Gly Tyr Asp Met 290 295 300 agg aaa ttg ctg aac ggt acg gtcgtt tcc aag cat ccg ttg aaa tcg 1380 Arg Lys Leu Leu Asn Gly Thr Val ValSer Lys His Pro Leu Lys Ser 305 310 315 320 gtt aca ttt gtc gat aac catgat aca cag ccg ggg caa tcg ctt gag 1428 Val Thr Phe Val Asp Asn His AspThr Gln Pro Gly Gln Ser Leu Glu 325 330 335 tcg act gtc caa aca tgg tttaag ccg ctt gct tac gct ttt att ctc 1476 Ser Thr Val Gln Thr Trp Phe LysPro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 aca agg gaa tct gga tac cctcag gtt ttc tac ggg gat atg tac ggg 1524 Thr Arg Glu Ser Gly Tyr Pro GlnVal Phe Tyr Gly Asp Met Tyr Gly 355 360 365 acg aaa gga gac tcc cag cgcgaa att cct gcc ttg aaa cac aaa att 1572 Thr Lys Gly Asp Ser Gln Arg GluIle Pro Ala Leu Lys His Lys Ile 370 375 380 gaa ccg atc tta aaa gcg agaaaa cag tat gcg tac gga gca cag cat 1620 Glu Pro Ile Leu Lys Ala Arg LysGln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 gat tat ttc gac cac catgac att gtc ggc tgg aca agg gaa ggc gac 1668 Asp Tyr Phe Asp His His AspIle Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 agc tcg gtt gca aat tcaggt ttg gcg gca tta ata aca gac gga ccc 1716 Ser Ser Val Ala Asn Ser GlyLeu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 ggt ggg gca aag cga atgtat gtc ggc cgg caa aac gcc ggt gag aca 1764 Gly Gly Ala Lys Arg Met TyrVal Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445 tgg cat gac att acc ggaaac cgt tcg gag ccg gtt gtc atc aat tcg 1812 Trp His Asp Ile Thr Gly AsnArg Ser Glu Pro Val Val Ile Asn Ser 450 455 460 gaa ggc tgg gga gag tttcac gta aac ggc ggg tcg gtt tca att tat 1860 Glu Gly Trp Gly Glu Phe HisVal Asn Gly Gly Ser Val Ser Ile Tyr 465 470 475 480 gtt caa aga tagaagagcagag aggacggatt tcctgaagga aatccgtttt 1912 Val Gln Arg tttatttt1920 8 483 PRT Bacillus licheniformis 8 Ala Asn Leu Asn Gly Thr Leu MetGln Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 Asn Asp Gly Gln His Trp ArgArg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30 Ala Glu His Gly Ile Thr AlaVal Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Ala Asp Val GlyTyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe His Gln Lys GlyThr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Gln Ser AlaIle Lys Ser Leu His Ser Arg Asp Ile Asn 85 90 95 Val Tyr Gly Asp Val ValIle Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110 Glu Asp Val Thr AlaVal Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125 Ile Ser Gly GluHis Leu Ile Lys Ala Trp Thr His Phe His Phe Pro 130 135 140 Gly Arg GlySer Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160 AspGly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185190 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195200 205 Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln210 215 220 Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe SerPhe 225 230 235 240 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr GlyLys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu GlyAla Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser ValPhe Asp Val Pro Leu 275 280 285 His Tyr Gln Phe His Ala Ala Ser Thr GlnGly Gly Gly Tyr Asp Met 290 295 300 Arg Lys Leu Leu Asn Gly Thr Val ValSer Lys His Pro Leu Lys Ser 305 310 315 320 Val Thr Phe Val Asp Asn HisAsp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr TrpPhe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser GlyTyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly AspSer Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380 Glu Pro IleLeu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 AspTyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425430 Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435440 445 Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser450 455 460 Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser IleTyr 465 470 475 480 Val Gln Arg 9 2084 DNA Bacillus amyloliquefaciensCDS (343)..(1794) BAN 9 gccccgcaca tacgaaaaga ctggctgaaa acattgagcctttgatgact gatgatttgg 60 ctgaagaagt ggatcgattg tttgagaaaa gaagaagaccataaaaatac cttgtctgtc 120 atcagacagg gtatttttta tgctgtccag actgtccgctgtgtaaaaat aaggaataaa 180 ggggggttgt tattatttta ctgatatgta aaatataatttgtataagaa aatgagaggg 240 agaggaaaca tgattcaaaa acgaaagcgg acagtttcgttcagacttgt gcttatgtgc 300 acgctgttat ttgtcagttt gccgattaca aaaacatcag ccgta aat ggc acg 354 Val Asn Gly Thr 1 ctg atg cag tat ttt gaa tgg tatacg ccg aac gac ggc cag cat tgg 402 Leu Met Gln Tyr Phe Glu Trp Tyr ThrPro Asn Asp Gly Gln His Trp 5 10 15 20 aaa cga ttg cag aat gat gcg gaacat tta tcg gat atc gga atc act 450 Lys Arg Leu Gln Asn Asp Ala Glu HisLeu Ser Asp Ile Gly Ile Thr 25 30 35 gcc gtc tgg att cct ccc gca tac aaagga ttg agc caa tcc gat aac 498 Ala Val Trp Ile Pro Pro Ala Tyr Lys GlyLeu Ser Gln Ser Asp Asn 40 45 50 gga tac gga cct tat gat ttg tat gat ttagga gaa ttc cag caa aaa 546 Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu GlyGlu Phe Gln Gln Lys 55 60 65 ggg acg gtc aga acg aaa tac ggc aca aaa tcagag ctt caa gat gcg 594 Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser GluLeu Gln Asp Ala 70 75 80 atc ggc tca ctg cat tcc cgg aac gtc caa gta tacgga gat gtg gtt 642 Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr GlyAsp Val Val 85 90 95 100 ttg aat cat aag gct ggt gct gat gca aca gaa gatgta act gcc gtc 690 Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp ValThr Ala Val 105 110 115 gaa gtc aat ccg gcc aat aga aat cag gaa act tcggag gaa tat caa 738 Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser GluGlu Tyr Gln 120 125 130 atc aaa gcg tgg acg gat ttt cgt ttt ccg ggc cgtgga aac acg tac 786 Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg GlyAsn Thr Tyr 135 140 145 agt gat ttt aaa tgg cat tgg tat cat ttc gac ggagcg gac tgg gat 834 Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly AlaAsp Trp Asp 150 155 160 gaa tcc cgg aag atc agc cgc atc ttt aag ttt cgtggg gaa gga aaa 882 Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg GlyGlu Gly Lys 165 170 175 180 gcg tgg gat tgg gaa gta tca agt gaa aac ggcaac tat gac tat tta 930 Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly AsnTyr Asp Tyr Leu 185 190 195 atg tat gct gat gtt gac tac gac cac cct gatgtc gtg gca gag aca 978 Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp ValVal Ala Glu Thr 200 205 210 aaa aaa tgg ggt atc tgg tat gcg aat gaa ctgtca tta gac ggc ttc 1026 Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu SerLeu Asp Gly Phe 215 220 225 cgt att gat gcc gcc aaa cat att aaa ttt tcattt ctg cgt gat tgg 1074 Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser PheLeu Arg Asp Trp 230 235 240 gtt cag gcg gtc aga cag gcg acg gga aaa gaaatg ttt acg gtt gcg 1122 Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu MetPhe Thr Val Ala 245 250 255 260 gag tat tgg cag aat aat gcc ggg aaa ctcgaa aac tac ttg aat aaa 1170 Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu GluAsn Tyr Leu Asn Lys 265 270 275 aca agc ttt aat caa tcc gtg ttt gat gttccg ctt cat ttc aat tta 1218 Thr Ser Phe Asn Gln Ser Val Phe Asp Val ProLeu His Phe Asn Leu 280 285 290 cag gcg gct tcc tca caa gga ggc gga tatgat atg agg cgt ttg ctg 1266 Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr AspMet Arg Arg Leu Leu 295 300 305 gac ggt acc gtt gtg tcc agg cat ccg gaaaag gcg gtt aca ttt gtt 1314 Asp Gly Thr Val Val Ser Arg His Pro Glu LysAla Val Thr Phe Val 310 315 320 gaa aat cat gac aca cag ccg gga cag tcattg gaa tcg aca gtc caa 1362 Glu Asn His Asp Thr Gln Pro Gly Gln Ser LeuGlu Ser Thr Val Gln 325 330 335 340 act tgg ttt aaa ccg ctt gca tac gccttt att ttg aca aga gaa tcc 1410 Thr Trp Phe Lys Pro Leu Ala Tyr Ala PheIle Leu Thr Arg Glu Ser 345 350 355 ggt tat cct cag gtg ttc tat ggg gatatg tac ggg aca aaa ggg aca 1458 Gly Tyr Pro Gln Val Phe Tyr Gly Asp MetTyr Gly Thr Lys Gly Thr 360 365 370 tcg cca aag gaa att ccc tca ctg aaagat aat ata gag ccg att tta 1506 Ser Pro Lys Glu Ile Pro Ser Leu Lys AspAsn Ile Glu Pro Ile Leu 375 380 385 aaa gcg cgt aag gag tac gca tac gggccc cag cac gat tat att gac 1554 Lys Ala Arg Lys Glu Tyr Ala Tyr Gly ProGln His Asp Tyr Ile Asp 390 395 400 cac ccg gat gtg atc gga tgg acg agggaa ggt gac agc tcc gcc gcc 1602 His Pro Asp Val Ile Gly Trp Thr Arg GluGly Asp Ser Ser Ala Ala 405 410 415 420 aaa tca ggt ttg gcc gct tta atcacg gac gga ccc ggc gga tca aag 1650 Lys Ser Gly Leu Ala Ala Leu Ile ThrAsp Gly Pro Gly Gly Ser Lys 425 430 435 cgg atg tat gcc ggc ctg aaa aatgcc ggc gag aca tgg tat gac ata 1698 Arg Met Tyr Ala Gly Leu Lys Asn AlaGly Glu Thr Trp Tyr Asp Ile 440 445 450 acg ggc aac cgt tca gat act gtaaaa atc gga tct gac ggc tgg gga 1746 Thr Gly Asn Arg Ser Asp Thr Val LysIle Gly Ser Asp Gly Trp Gly 455 460 465 gag ttt cat gta aac gat ggg tccgtc tcc att tat gtt cag aaa taa 1794 Glu Phe His Val Asn Asp Gly Ser ValSer Ile Tyr Val Gln Lys 470 475 480 ggtaataaaa aaacacctcc aagctgagtgcgggtatcag cttggaggtg cgtttatttt 1854 ttcagccgta tgacaaggtc ggcatcaggtgtgacaaata cggtatgctg gctgtcatag 1914 gtgacaaatc cgggttttgc gccgtttggctttttcacat gtctgatttt tgtataatca 1974 acaggcacgg agccggaatc tttcgccttggaaaaataag cggcgatcgt agctgcttcc 2034 aatatggatt gttcatcggg atcgctgcttttaatcacaa cgtgggatcc 2084 10 483 PRT Bacillus amyloliquefaciens 10 ValAsn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp 1 5 10 15Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20 25 30Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser 35 40 45Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu 50 55 60Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu 65 70 7580 Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr 85 9095 Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp 100105 110 Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser115 120 125 Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro GlyArg 130 135 140 Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His PheAsp Gly 145 150 155 160 Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg IlePhe Lys Phe Arg 165 170 175 Gly Glu Gly Lys Ala Trp Asp Trp Glu Val SerSer Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Val AspTyr Asp His Pro Asp Val 195 200 205 Val Ala Glu Thr Lys Lys Trp Gly IleTrp Tyr Ala Asn Glu Leu Ser 210 215 220 Leu Asp Gly Phe Arg Ile Asp AlaAla Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val GlnAla Val Arg Gln Ala Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala GluTyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn 260 265 270 Tyr Leu Asn LysThr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu 275 280 285 His Phe AsnLeu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg ArgLeu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala 305 310 315 320Val Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330335 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340345 350 Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly355 360 365 Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp AsnIle 370 375 380 Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly ProGln His 385 390 395 400 Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp ThrArg Glu Gly Asp 405 410 415 Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala LeuIle Thr Asp Gly Pro 420 425 430 Gly Gly Ser Lys Arg Met Tyr Ala Gly LeuLys Asn Ala Gly Glu Thr 435 440 445 Trp Tyr Asp Ile Thr Gly Asn Arg SerAsp Thr Val Lys Ile Gly Ser 450 455 460 Asp Gly Trp Gly Glu Phe His ValAsn Asp Gly Ser Val Ser Ile Tyr 465 470 475 480 Val Gln Lys 11 1458 DNABacillus sp. CDS (1)..(1458) AA560 11 cac cat aat ggt acg aac ggc acaatg atg cag tac ttt gaa tgg tat 48 His His Asn Gly Thr Asn Gly Thr MetMet Gln Tyr Phe Glu Trp Tyr 1 5 10 15 cta cca aat gac gga aac cat tggaat aga tta agg tct gat gca agt 96 Leu Pro Asn Asp Gly Asn His Trp AsnArg Leu Arg Ser Asp Ala Ser 20 25 30 aac cta aaa gat aaa ggg atc tca gcggtt tgg att cct cct gca tgg 144 Asn Leu Lys Asp Lys Gly Ile Ser Ala ValTrp Ile Pro Pro Ala Trp 35 40 45 aag ggt gcc tct caa aat gat gtg ggg tatggt gct tat gat ctg tat 192 Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr GlyAla Tyr Asp Leu Tyr 50 55 60 gat tta gga gaa ttc aat caa aaa gga acc attcgt aca aaa tat gga 240 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile ArgThr Lys Tyr Gly 65 70 75 80 acg cgc aat cag tta caa gct gca gtt aac gccttg aaa agt aat gga 288 Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala LeuLys Ser Asn Gly 85 90 95 att caa gtg tat ggc gat gtt gta atg aat cat aaaggg gga gca gac 336 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys GlyGly Ala Asp 100 105 110 gct acc gaa atg gtt agg gca gtt gaa gta aac ccgaat aat aga aat 384 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro AsnAsn Arg Asn 115 120 125 caa gaa gtg tcc ggt gaa tat aca att gag gct tggaca aag ttt gac 432 Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp ThrLys Phe Asp 130 135 140 ttt cca gga cga ggt aat act cat tca aac ttc aaatgg aga tgg tat 480 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys TrpArg Trp Tyr 145 150 155 160 cac ttt gat gga gta gat tgg gat cag tca cgtaag ctg aac aat cga 528 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg LysLeu Asn Asn Arg 165 170 175 att tat aaa ttt aga ggt gat gga aaa ggg tgggat tgg gaa gtc gat 576 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp AspTrp Glu Val Asp 180 185 190 aca gaa aac ggt aac tat gat tac cta atg tatgca gat att gac atg 624 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr AlaAsp Ile Asp Met 195 200 205 gat cac cca gag gta gtg aat gag cta aga aattgg ggt gtt tgg tat 672 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn TrpGly Val Trp Tyr 210 215 220 acg aat aca tta ggc ctt gat ggt ttt aga atagat gca gta aaa cat 720 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile AspAla Val Lys His 225 230 235 240 ata aaa tac agc ttt act cgt gat tgg attaat cat gtt aga agt gca 768 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile AsnHis Val Arg Ser Ala 245 250 255 act ggc aaa aat atg ttt gcg gtt gcg gaattt tgg aaa aat gat tta 816 Thr Gly Lys Asn Met Phe Ala Val Ala Glu PheTrp Lys Asn Asp Leu 260 265 270 ggt gct att gaa aac tat tta aac aaa acaaac tgg aac cat tca gtc 864 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr AsnTrp Asn His Ser Val 275 280 285 ttt gat gtt ccg ctg cac tat aac ctc tataat gct tca aaa agc gga 912 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr AsnAla Ser Lys Ser Gly 290 295 300 ggg aat tat gat atg agg caa ata ttt aatggt aca gtc gtg caa aga 960 Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn GlyThr Val Val Gln Arg 305 310 315 320 cat cca atg cat gct gtt aca ttt gttgat aat cat gat tcg caa cct 1008 His Pro Met His Ala Val Thr Phe Val AspAsn His Asp Ser Gln Pro 325 330 335 gaa gaa gct tta gag tct ttt gtt gaagaa tgg ttc aaa cca tta gcg 1056 Glu Glu Ala Leu Glu Ser Phe Val Glu GluTrp Phe Lys Pro Leu Ala 340 345 350 tat gct ttg aca tta aca cgt gaa caaggc tac cct tct gta ttt tat 1104 Tyr Ala Leu Thr Leu Thr Arg Glu Gln GlyTyr Pro Ser Val Phe Tyr 355 360 365 gga gat tat tat ggc att cca acg catggt gta cca gcg atg aaa tcg 1152 Gly Asp Tyr Tyr Gly Ile Pro Thr His GlyVal Pro Ala Met Lys Ser 370 375 380 aaa att gac ccg att cta gaa gcg cgtcaa aag tat gca tat gga aga 1200 Lys Ile Asp Pro Ile Leu Glu Ala Arg GlnLys Tyr Ala Tyr Gly Arg 385 390 395 400 caa aat gac tac tta gac cat cataat atc atc ggt tgg aca cgt gaa 1248 Gln Asn Asp Tyr Leu Asp His His AsnIle Ile Gly Trp Thr Arg Glu 405 410 415 ggg aat aca gca cac ccc aac tccggt tta gct act atc atg tcc gat 1296 Gly Asn Thr Ala His Pro Asn Ser GlyLeu Ala Thr Ile Met Ser Asp 420 425 430 ggg gca gga gga aat aag tgg atgttt gtt ggg cgt aat aaa gct ggt 1344 Gly Ala Gly Gly Asn Lys Trp Met PheVal Gly Arg Asn Lys Ala Gly 435 440 445 caa gtt tgg acc gat atc act ggaaat cgt gca ggt act gtt acg att 1392 Gln Val Trp Thr Asp Ile Thr Gly AsnArg Ala Gly Thr Val Thr Ile 450 455 460 aat gct gat gga tgg ggt aat ttttct gta aat gga gga tca gtt tct 1440 Asn Ala Asp Gly Trp Gly Asn Phe SerVal Asn Gly Gly Ser Val Ser 465 470 475 480 att tgg gta aac aaa taa 1458Ile Trp Val Asn Lys 485 12 485 PRT Bacillus sp. 12 His His Asn Gly ThrAsn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn AspGly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30 Asn Leu Lys AspLys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Ala SerGln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly GluPhe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg AsnGln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95 Ile Gln ValTyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala ThrGlu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 GlnGlu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg165 170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu ValAsp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp IleAsp Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp GlyVal Trp Tyr 210 215 220 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile AspAla Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp IleAsn His Val Arg Ser Ala 245 250 255 Thr Gly Lys Asn Met Phe Ala Val AlaGlu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr Leu AsnLys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His TyrAsn Leu Tyr Asn Ala Ser Lys Ser Gly 290 295 300 Gly Asn Tyr Asp Met ArgGln Ile Phe Asn Gly Thr Val Val Gln Arg 305 310 315 320 His Pro Met HisAla Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Glu Glu AlaLeu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr AlaLeu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 GlyAsp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg 385 390395 400 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu405 410 415 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met SerAsp 420 425 430 Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn LysAla Gly 435 440 445 Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly ThrVal Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn GlyGly Ser Val Ser 465 470 475 480 Ile Trp Val Asn Lys 485 13 485 PRTBacillus 707 13 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe GluTrp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn SerAsp Ala Ser 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile ProPro Ala Trp 35 40 45 Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala TyrAsp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg ThrLys Tyr Gly 65 70 75 80 Thr Arg Ser Gln Leu Gln Ala Ala Val Thr Ser LeuLys Asn Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His LysGly Gly Ala Asp 100 105 110 Ala Thr Glu Met Val Arg Ala Val Glu Val AsnPro Asn Asn Arg Asn 115 120 125 Gln Glu Val Thr Gly Glu Tyr Thr Ile GluAla Trp Thr Arg Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His SerSer Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp TrpAsp Gln Ser Arg Arg Leu Asn Asn Arg 165 170 175 Ile Tyr Lys Phe Arg GlyHis Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly AsnTyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 Asp His Pro GluVal Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn ThrLeu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 IleLys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245 250 255Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265270 Gly Ala Ile Glu Asn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser Val 275280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly290 295 300 Gly Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val GlnArg 305 310 315 320 His Pro Ser His Ala Val Thr Phe Val Asp Asn His AspSer Gln Pro 325 330 335 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp PheLys Pro Leu Ala 340 345 350 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly TyrPro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His GlyVal Pro Ala Met Arg Ser 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala ArgGln Lys Tyr Ala Tyr Gly Lys 385 390 395 400 Gln Asn Asp Tyr Leu Asp HisHis Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Thr Ala His ProAsn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Ala Gly Gly SerLys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435 440 445 Gln Val Trp SerAsp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 Asn Ala AspGly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 IleTrp Val Asn Lys 485 14 20 DNA Artificial Sequence misc_feature (1)..(20)Forward Primer 14 gacctgcagt caggcaacta 20 15 20 DNA Artificial Sequencemisc_feature (1)..(20) Reverse Primer 15 tagagtcgac ctgcaggcat 20

1. A alpha-amylase variant of a parent Termamyl-like alpha-amylase,comprising an alteration at one or more of the following regions orpositions selected from the group consisting of: 186-193, 261-276,283-293, 334-339,and 234, wherein each region or position corresponds toa region or position of the amino acid sequence of B. licheniformisalpha-amylase shown in seq id no:
 8. 2. The variant of claim 1, whereinthe variant has an alteration in one or more of the following positions:W263,E189,L335,Y290,N265,V286,Q264,K234. 3.The variant of claim 1,wherein the variant has the following alterations: W263GASTV; E189GASTV,L335GASTV, Y290 A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,V;N265G,A,S,T,V;V286F,W,Y,G,A,S; Q264X, K234X, preferably NQ.
 4. The variant of any ofclaims 1-3, wherein the parent Termamyl-like alpha-amylase is derivedfrom a strain of B. licheniformis, B. amyloliquefaciens, B.stearothermophilus, Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 orDSM 9375, or DSMZ no. 12649, KSM AP1378.
 5. The variant of claims 1-4,wherein the parent Termamyl-like alpha-amylase is any of thealpha-amylases selected from the group depicted in SEQ ID NOS: 2, 4, 6,8, 10, 12, and
 13. 6. The variant according to any of claims 1-5,wherein the parent Termamyl-like alpha-amylase has an amino acidsequence which has a degree of identity to SEQ ID NO: 4 of at least 60%.7. The variant of any of claims 1-6, wherein the parent Termamyl-likealpha-amylase is encoded by a nucleic acid sequence which hydridizesunder low stringency conditions with the nucleic acid sequence of SEQ IDNO:
 7. 8. The variant of claims 1-7, which variant has alteredalpha-1,6-D-glucosidic branch linkage cleavage activity on amylopectin,referably increased alpha-1,6-D-glucosidic branch linkage cleavageactivity of amylopectin or a limit dextrin prepared by TERMAMYL™ orNOVAMYL®.
 9. A DNA construct comprising a DNA sequence encoding analpha-amylase variant according to any one of claims 1 to
 8. 10. Arecombinant expression vector which carries a DNA construct according toclaim
 9. 11. A cell which is transformed with a DNA construct accordingto claim 9 or a vector according to claim
 10. 12. A cell according toclaim 11, which is a microorganism, preferably a bacterium or a fungus.13. The cell according to claim 12, which cell is a gram positivebacterium, such as Bacillus subtilis, Bacillus licheniformis, Bacilluslentus, Bacillus brevis, Bacillus stearothermophilus, Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus coagu-lans, Bacilluscirculans, Bacillus lautus or Bacillus thu-ringiensis.
 14. A detergentadditive comprising an alpha-amylase variant according to any one ofclaims 1 to 8, optionally in the form of a non-dusting granulate,stabilised liquid or protected enzyme.
 15. A detergent additiveaccording to claim 14, which contains 0.02-200 mg of enzyme protein/g ofthe additive.
 16. A detergent additive according to claims 14 or 15,which additionally comprises another enzyme such as a protease, alipase, a peroxidase, amylase or another amylolytic enzyme, such asglucoamylase, and/or a cellulase.
 17. A detergent composition comprisingan alpha-amylase variant according to any of claims 1 to
 8. 18. Adetergent composition according to claim 17, which additionallycomprises another enzyme such as a protease, a lipase, a peroxidase,another amylolytic enzyme and/or a cellulase.
 19. A manual or automaticdishwashing detergent composition comprising an alpha-amylase variantaccording to any of claims 1 to
 8. 20. A dishwashing detergentcomposition according to claim 19, which additionally comprises anotherenzyme such as a protease, a lipase, a peroxidase, amylase or anotheramylolytic enzyme, such as glucoamylase, and/or a cellulase.
 21. Amanual or automatic laundry washing composition comprising analpha-amylase variant according to any of claims 1 to
 8. 22. A laundrywashing composition according to claim 21, which additionally comprisesanother enzyme such as a protease, a lipase, a peroxidase, an amylaseand/or another an amylolytic enzyme, such as glucoamylase and/or acellulase.
 23. A composition comprising an alpha-amylase variant of anyis of claims 1-8.
 24. The composition of claims 23, which furthercomprise another alpha-amylase, glucoamylase, pullulanase, isoamylase,protease, preferably acidic protease, especially from Aspergillus, suchas A. niger or A. aculatus.